Anti-amyloid beta antibodies binding to a cyclic amyloid beta peptide

ABSTRACT

The disclosure pertains to epitopes identified in A-beta including conformational epitopes, antibodies thereto and methods of making and using immunogens and antibodies specific thereto.

RELATED APPLICATIONS

This application is a national phase entry of PCT/CA2016/051305, filedNov. 9, 2016, which claims the benefit of priority of U.S. PatentApplication Ser. No. 62/253,044, filed Nov. 9, 2015; U.S. PatentApplication Ser. No. 62/289,893, filed on Feb. 1, 2016; U.S. PatentApplication Ser. No. 62/365,634, filed on Jul. 22, 2016; and U.S. PatentApplication Ser. No. 62/393,615, filed on Sep. 12, 2016, each of whichare incorporated herein by reference.

FIELD

The present disclosure relates to Amyloid beta (A-beta or Aβ) epitopesand antibodies thereto and more specifically to conformational A-betaepitopes that are predicted to be selectively accessible in A-betaoligomers, and related antibody compositions and uses thereof.

BACKGROUND

Amyloid-beta (A-beta), which exists as a 36-43 amino acid peptide, is aproduct released from amyloid precursor protein (APP) by the enzymes βand γ secretase. In AD patients, A-beta can be present in solublemonomers, insoluble fibrils and soluble oligomers. In monomer form,A-beta exists as a predominantly unstructured polypeptide chain. Infibril form, A-beta can aggregate into distinct morphologies, oftenreferred to as strains. Several of these structures have been determinedby solid-state NMR.

For, example, structures for several stains of fibrils are available inthe Protein Data Bank (PDB), a crystallographic database of atomicresolution three dimensional structural data, including a 3-foldsymmetric Aβ structure (PDB entry, 2M4J); a two-fold symmetric structureof Aβ-40 monomers (PDB entry 2LMN), and a single-chain, parallelin-register structure of Aβ-42 monomers (PDB entry 2MXU).

The structure of 2M4J is reported in J. X. LU, W. QIANG, W. M. YAU, C.D. SCHWIETERS, S. C. MEREDITH, R. TYCKO, MOLECULAR STRUCTURE OFBETA-AMYLOID FIBRILS IN ALZHEIMER'S DISEASE BRAIN TISSUE. CELL Vol. 154p. 1257 (2013) and the structure of 2MXU is reported in Y. XIAO, B. MA,D. MCELHENY, S. PARTHASARATHY, F. LONG, M. HOSHI, R. NUSSINOV, Y. ISHIIA BETA (1-42) FIBRIL STRUCTURE ILLUMINATES SELF-RECOGNITION ANDREPLICATION OF AMYLOID IN ALZHEIMER'S DISEASE. NAT. STRUCT. MOL. BIOL.Vol. 22 p. 499 (2015).

A-beta oligomers have been shown to kill cell lines and neurons inculture and block a critical synaptic activity that subserves memory,referred to as long term potentiation (LTP), in slice cultures andliving animals.

The structure of the oligomer has not been determined to date. Moreover,NMR and other evidence indicates that the oligomer exists not in asingle well-defined structure, but in a conformationally-plastic,malleable structural ensemble with limited regularity. Moreover, theconcentration of toxic oligomer species is far below either that of themonomer or fibril (estimates vary but on the order of 1000-fold below ormore), making this target elusive.

U.S. Pat. Nos. 5,766,846; 5,837,672; and 5,593,846 (which areincorporated herein by reference) describe the production of murinemonoclonal antibodies to the central domain of the Aβ peptide. WO01/62801 describes antibodies that bind A-beta between amino acids13-28. WO2004071408 discloses humanized antibodies. WO2008088983A1describes an antibody fragment that binds amyloid beta (A-beta) peptideand is covalently attached to one or more molecules of polyethyleneglycol (PEG), the antibody fragment specifically binding human A-betapeptide between amino acid positions 13-28. Solanezumab and Crenezumabbind amino acids 16-26 on A-beta. Crenezumab interacts with the monomer,oligomer and fibril. Midregion antibodies, including solanezumab(picomolar affinity) and crenezumab (nanomolar affinity), appear topreferentially bind monomeric A-beta [1].

Antibodies that preferentially or selectively bind A-beta oligomers aredesirable.

SUMMARY

Described herein is an epitope, optionally a conformational epitope, inA-beta consisting of residues QKLV (SEQ ID NO: 1) or a part thereof, andantibodies thereto. The epitope is identified as selectively exposed inthe oligomeric species of A-beta, in a conformation that distinguishesit from that in the monomer.

An aspect includes a compound, preferably a cyclic compound comprisingan A-beta peptide the peptide comprising QKL and up to 8, 7 or 6 A-betaresidues, and a linker, wherein the linker is covalently coupled to theA-beta peptide N-terminus residue and the A-beta C-terminus residue.

In an embodiment, the A-beta peptide is selected from a peptide having asequence of any one of SEQ ID NOS: 1-10, optionally selected from QKLV(SEQ ID NO: 1), HQKLV (SEQ ID NO: 2), HQKLVF (SEQ ID NO: 9) and QKLVF(SEQ ID NO: 10).

In another embodiment, the cyclic compound is a cyclic peptide.

In another embodiment, the cyclic compound comprises i) at least Q in analternate conformation compared to Q in the context of a correspondinglinear compound and/or ii) a conformation for Q, and/or K, and/or L asmeasured by entropy that is at least 10%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40% more constrained compared to acorresponding linear compound.

In another embodiment, the A-beta peptide is QKLV (SEQ ID NO: 1).

In another embodiment, the compound further comprises a detectablelabel.

In another embodiment, the linker comprises or consists of 1-8 aminoacids and/or equivalently functioning molecules and/or one or morefunctionalizable moieties.

In another embodiment, the linker amino acids are selected from A and G,and/or wherein the functionalizable moiety is C.

In another embodiment, the linker comprises or consists of amino acidsGCG or CGC.

In another embodiment, the linker comprises a PEG molecule.

In another embodiment, the cyclic compound is selected from thefollowing structures:

and

An aspect includes an immunogen comprising the cyclic compound describedherein.

In an embodiment, the cyclic compound is coupled to a carrier protein orimmunogenicity enhancing agent.

In an embodiment, the carrier protein is bovine serum albumin (BSA) orthe immunogenicity-enhancing agent is keyhole limpet haemocyanin (KLH).

An aspect includes a composition comprising the compound describedherein or the immunogen described herein.

In an embodiment, the composition described herein, further comprises anadjuvant.

In another embodiment, the adjuvant is aluminum phosphate or aluminumhydroxide.

An aspect includes isolated antibody that specifically binds to anA-beta peptide having a sequence of QKLV (SEQ ID NO: 1) or a relatedepitope sequence, optionally as set forth in any one of SEQ ID NOS:1-10.

In an embodiment, the antibody specifically binds an epitope on A-beta,wherein the epitope comprises at least two consecutive amino acidresidues predominantly involved in binding to the antibody, wherein theat least two consecutive amino acids are QK embedded within QKLV (SEQ IDNO: 1).

In another embodiment, the epitope comprises or consists of QKLV (SEQ IDNO: 1), HQKLV (SEQ ID NO: 2), HQKLVF (SEQ ID NO: 9) or QKLVF (SEQ ID NO:10).

In another embodiment, the antibody is a conformation specific and/orselective antibody that specifically or selectively binds to AEDV or arelated epitope peptide presented in a cyclic compound, optionally acyclic compound described herein, preferably a cyclic peptide having asequence as set forth in SEQ ID NO: 3.

In another embodiment, the antibody selectively binds A-beta oligomerover A-beta monomer and/or A-beta fibril.

In another embodiment, the selectivity is at least 3 fold, at least 5fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40fold, at least 50 fold, at least 100 fold, at least 500 fold, at least1000 fold more selective for A-beta oligomer over A-beta monomer and/orA-beta fibril.

In another embodiment, the antibody does not specifically and/orselectively bind a linear peptide comprising sequence QKLV (SEQ IDNO: 1) or a related epitope, optionally wherein the sequence of thelinear peptide is a linear version of a cyclic compound used to raisethe antibody, optionally a linear peptide having a sequence as set forthin SEQ ID NO: 3.

In another embodiment, the antibody lacks or has negligible binding toA-beta monomer and/or A-beta fibril plaques in situ.

In an embodiment, the antibody is produced using a cyclic compounddescribed herein, optionally a cyclic peptide described herein.

In another embodiment, the antibody is a monoclonal antibody or apolyclonal antibody.

In another embodiment, the antibody is a humanized antibody.

In another embodiment, the antibody is an antibody binding fragmentselected from Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers,nanobodies, minibodies, diabodies, and multimers thereof.

In another embodiment, the antibody described herein, comprises a lightchain variable region and a heavy chain variable region, optionallyfused, the heavy chain variable region comprising complementaritydetermining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variableregion comprising complementarity determining region CDR-L1, CDR-L2 andCDR-L3 and with the amino acid sequences of said CDRs comprising thesequences:

CDR-H1 (SEQ ID NO: 11) GYTFTDYE CDR-H2 (SEQ ID NO: 12) IDPETGDT CDR-H3(SEQ ID NO: 13) TSPIYYDYDWFAY CDR-L1 (SEQ ID NO: 14) QSLLNNRTRKNY CDR-L2(SEQ ID NO: 15) WAS CDR-L3 (SEQ ID NO: 16) KQSYNLRT CDR-H1(SEQ ID NO: 21) GFSLSTSGMG CDR-H2 (SEQ ID NO: 22) IWWDDDK CDR-H3(SEQ ID NO: 23) ARSITTVVATPFDY CDR-L1 (SEQ ID NO: 24) QNVRSA CDR-L2(SEQ ID NO: 25) LAS CDR-L3 (SEQ ID NO: 26) LQHWNSPFT

In another embodiment, the antibody comprises a heavy chain variableregion comprising: i) an amino acid sequence as set forth in SEQ ID NO:18 or 28; ii) an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% sequence identity to SEQ ID NO:18 or 28, wherein the CDR sequences are as set forth in SEQ ID NO:11,12, 13, 21, 22 and 23, or iii) a conservatively substituted amino acidsequence i).

In another embodiment, the antibody comprises a light chain variableregion comprising i) an amino acid sequence as set forth in SEQ ID NO:20 or 30, ii) an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80% or at least 90% sequence identity to SEQ ID NO:20 or 30, wherein the CDR sequences are as set forth in SEQ ID NO: 14,15, 16, 24, 25 and 26, or iii) a conservatively substituted amino acidsequence of i).

In another embodiment, the heavy chain variable region amino acidsequence is encoded by a nucleotide sequence as set forth in SEQ ID NO:17 or 27 or a codon degenerate or optimized version thereof; and/or theantibody comprises a light chain variable region amino acid sequenceencoded by a nucleotide sequence as set out in SEQ ID NO: 19 or 29 or acodon degenerate or optimized version thereof.

In another embodiment, the heavy chain variable region comprises orconsists of an amino acid sequence as set forth in SEQ ID NO:18 or 28and/or the light chain variable region comprises or consists of an aminoacid sequence as set forth in SEQ ID NO: 20 or 30.

In another embodiment, the antibody competes for binding to human A-betawith an antibody comprising the CDR sequences as recited in Table 6and/or 8.

An aspect includes an immunoconjugate comprising the antibody describedherein and a detectable label or cytotoxic agent.

In an embodiment, the detectable label comprises a positron emittingradionuclide, optionally for use in subject imaging such as PET imaging.

An aspect includes a composition comprising the antibody describedherein, or the immunoconjugate described herein, optionally with adiluent.

An aspect includes a nucleic acid molecule encoding a proteinaceousportion of the compound or immunogen described herein, the antibodydescribed herein or proteinaceous immunoconjugates described herein.

An aspect includes a vector comprising the nucleic acid describedherein.

An aspect includes a cell expressing an antibody described herein,optionally wherein the cell is a hybridoma comprising the vectordescribed herein.

An aspect includes a kit comprising the compound described herein, theimmunogen described herein, the antibody described herein, theimmunoconjugate described herein, the composition described herein, thenucleic acid molecule described herein, the vector described herein orthe cell described herein.

An aspect includes a method of making the antibody described herein,comprising administering the compound or immunogen described herein or acomposition comprising said compound or immunogen to a subject andisolating antibody and/or cells expressing antibody specific orselective for the compound or immunogen administered and/or A-betaoligomers, optionally lacking or having negligible binding to a linearpeptide comprising the A-beta peptide and/or lacking or havingnegligible plaque binding.

An aspect includes a method of determining if a biological samplecomprises A-beta, the method comprising:

-   -   a. contacting the biological sample with an antibody described        herein or the immunoconjugate described herein; and    -   b. detecting the presence of any antibody complex.

In an embodiment, the biological sample contains A-beta oligomer themethod comprising:

-   -   a. contacting the sample with the antibody described herein or        the immunoconjugate described herein that is specific and/or        selective for A-beta oligomers under conditions permissive for        forming an antibody: A-beta oligomer complex; and    -   b. detecting the presence of any complex;    -   wherein the presence of detectable complex is indicative that        the sample may contain A-beta oligomer.

In another embodiment, the amount of complex is measured.

In another embodiment, the sample comprises brain tissue or an extractthereof, whole blood, plasma, serum and/or CSF.

In another embodiment, the sample is a human sample.

In another embodiment, the sample is compared to a control, optionally aprevious sample.

In another embodiment, the level of A-beta is detected by SPR.

An aspect includes a method of measuring a level of A-beta in a subject,the method comprising administering to a subject at risk or suspected ofhaving or having AD, an immunoconjugate comprising an antibody describedherein wherein the antibody is conjugated to a detectable label; anddetecting the label, optionally quantitatively detecting the label.

In an embodiment, the label is a positron emitting radionuclide.

An aspect includes a method of inducing an immune response in a subject,comprising administering to the subject a compound or combination ofcompounds described herein, optionally a cyclic compound comprising QKLV(SEQ ID NO:1) or a related epitope peptide sequence, an immunogen and/orcomposition comprising said compound or said immunogen; and optionallyisolating cells and/or antibodies that specifically or selectively bindthe A-beta peptide in the compound or immunogen administered.

An aspect includes a method of inhibiting A-beta oligomer propagation,the method comprising contacting a cell or tissue expressing A-beta withor administering to a subject in need thereof an effective amount of anA-beta oligomer specific or selective antibody or immunoconjugatedescribed herein, to inhibit A-beta aggregation and/or oligomerpropagation.

An aspect includes a method of treating AD and/or other A-beta amyloidrelated diseases, the method comprising administering to a subject inneed thereof i) an effective amount of an antibody or immunoconjugatedescribed herein, optionally an A-beta oligomer specific or selectiveantibody, or a pharmaceutical composition comprising said antibody; 2)administering an isolated cyclic compound comprising QKLV (SEQ ID NO:1)or a related epitope sequence or immunogen or pharmaceutical compositioncomprising said cyclic compound, or 3) a nucleic acid or vectorcomprising a nucleic acid encoding the antibody of 1 or the immunogen of2, to a subject in need thereof.

In an embodiment, a biological sample from the subject to be treated isassessed for the presence or levels of A-beta using an antibodydescribed herein.

In another embodiment, more than one antibody or immunogen isadministered.

In another embodiment, the antibody, immunoconjugate, immunogen,composition or nucleic acid or vector is administered directly to thebrain or other portion of the CNS.

In another embodiment, the composition is a pharmaceutical compositioncomprising the compound or immunogen in admixture with apharmaceutically acceptable, diluent or carrier.

An aspect includes an isolated peptide comprising an A beta peptideconsisting of the sequence of any one of the sequences set forth in SEQID NOS: 1-10.

In an embodiment, the peptide is a cyclic peptide comprising a linkerwherein the linker is covalently coupled to the A-beta peptideN-terminus residue and/or the A-beta C-terminus residue.

In another embodiment, the isolated peptide described herein comprises adetectable label.

An aspect includes a nucleic acid sequence encoding the isolated peptidedescribed herein.

An aspect includes a hybridoma expressing the antibody described herein.

Other features and advantages of the present disclosure will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the disclosure aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the present disclosure will now be described inrelation to the drawings in which:

FIG. 1 is a free energy landscape graph depicting the partial unfoldingof 2M4J.

FIG. 2 is a landscape graph of partially unfolding 2MXU, threshold 7kcal/mol.

FIG. 3 is a schematic showing curvature as a function of residue index.The long arrow identifies the cyclic peptide and the short arrowidentifies the linear peptide.

FIG. 4(a) is a series of graphs showing the dihedral angledistributions, for all the dihedral angles involving the side chainheavy atoms of residue Q15, and FIG. 4(b) is a schematic of a cyclicpeptide comprising QKLV (SEQ ID NO: 1).

FIG. 5 is a graph showing dihedral angle distributions for anglesinvolving the side chain heavy atoms of K16.

FIG. 6 is a graph showing dihedral angle distributions for anglesinvolving the side chain heavy atoms of L17.

FIG. 7 is a graph showing dihedral angle distributions for anglesinvolving the side chain heavy atoms of V18.

FIG. 8 is a graph showing the likelihood of exposure as a function ofsequence, as determined by the collective coordinates method. A peakemerges around centered residue 17.

FIG. 9 is a graph showing the solubility vs residue index for A-beta 42peptide.

FIG. 10 is a series of plots of the solvent accessible surface area(SASA), the weighted SASA, σ_(i)·SASA_(i), and σ_(i)·SASA_(i)−(σ_(i) 19SASA_(i))_(fibral). An arrow identifies the cyclic structure in eachpanel.

FIG. 11 shows plots of the SASA of heavy atom-hydrogen moieties alongthe side chains of residues QKLV (SEQ ID NO: 1). An arrow identifies thecyclic structure in each panel.

FIG. 12 is a schematic showing a series of cyclic compounds comprisingQKLV (SEQ ID NO: 1).

FIG. 13 is a graph showing the change in entropy for each of residues ofQKLV (SEQ ID NO: 1), in the cyclic peptide Cyclo(CGQKLVG) (SEQ ID NO: 3)and linear peptide CGQKLVG (SEQ ID NO: 3), both as compared to thecorresponding entropy of those residues in the fibril.

FIG. 14 is a schematic showing that the dihedral angle between theCα-Cβ-Cγ-Cδ atoms in the side chain of Q is different between the cyclicpeptide and the linear peptide.

FIG. 15: Surface plasmon resonance (SPR) direct binding assay of tissueculture supernatant clones to cyclic peptide and linear peptide in PanelA, and A-beta oligomer and A-beta monomer in Panel B.

FIG. 16: Plot comparing tissue culture supernatant clone binding in SPRdirect binding assay versus ELISA.

FIG. 17: SPR direct binding assay of select clones to cyclic peptide,linear peptide, A-beta monomer, and A-beta oligomer.

FIG. 18: Immunohistochemical staining of plaque from cadaveric AD brainusing 6E10 positive control antibody (A) and a selected and purifiedantibody (305-62, 8H10) raised against cyclo(CGQKLVG) (SEQ ID NO: 3)(B).

FIG. 19: Secondary screening of selected and purified antibodies usingan SPR indirect (capture) binding assay. SPR binding response of A-betaoligomer to captured antibody minus binding response of A-beta monomerto captured antibody (circle); SPR binding response of pooled solublebrain extract (BH) from AD patients to captured antibody minus bindingresponse of pooled brain extract from non-AD controls to capturedantibody (triangle); SPR binding response of pooled cerebrospinal fluid(CSF) from AD patients to captured antibody minus binding response ofpooled CSF from non-AD controls to captured antibody (square).

FIG. 20: Verification of antibody binding to stable A-beta oligomers.SPR sensorgrams and binding response plots of varying concentrations ofcommercially-prepared stable A-beta oligomers binding to immobilizedantibodies. Panel A shows results with the positive control mAb6E10,Panel B with the negative isotype control and Panel C with antibodyraised against cyclo (CGQKLVG) (SEQ ID NO: 3). Panel D plots binding ofselected antibody clones raised against cyclic peptide comprising QKLV(SEQ ID No: 1), with A-beta oligomer at a concentration of 1 micromolar.

FIG. 21 A-C: Plots showing propagation of A-beta aggregation in vitro inthe presence (stars) or absence (squares) of representative antibodiesraised using a cyclic peptide comprising QKLV (SEQ ID NO: 1).

FIG. 22 A-B: A) A plot showing the viability of rat primary corticalneurons exposed to toxic A-beta oligomers (AβO) in the presence orabsence of different molar ratios of a negative isotype control (A) oran antibody (B) raised against cyclo(CGQKLVG (SEQ ID NO: 3). Controlsinclude neurons cultured alone (CTRL), neurons incubated with antibodywithout oligomers and neurons cultured with the neuroprotective humaninpeptide (HNG) with or without Aβ oligomers

Table 1 shows the binding properties of selected tissue culturesupernatant clones.

Table 2 shows the binding properties summary for selected purifiedantibodies.

Table 3 lists the oligomer binding—monomer binding for an antibodyraised against cyclo(CGQKLVG) (SEQ ID NO: 3).

Table 4 lists properties of antibodies tested on formalin fixed tissues.

Table 5 is an exemplary toxicity assay.

Table 6 lists CDR sequences of clone 305-61 (7E9)

Table 7 lists heavy chain and light chain variable sequences of clone305-61 (7E9)

Table 8 lists CDR sequences of clone 305-62 (8H10)

Table 9 lists heavy chain and light chain variable sequences of clone305-62 (8H10)

Table 10 is a table of A-beta epitope sequences and select A-betasequences with linker.

Table 11 provides the full A-beta 1-42 human polypeptide sequence

DETAILED DESCRIPTION OF THE DISCLOSURE

A prerequisite for the generation of oligomer-specific antibodies is theidentification of targets on A-beta peptide that are not present, orpresent to a much lesser degree, on either the monomer or fibril. Theseoligomer-specific epitopes may not differ in primary sequence from thecorresponding segment in monomer or fibril, however they would beconformationally distinct in the context of the oligomer. That is, theywould present a distinct conformation in terms of backbone and/orsidechain conformation in the oligomer that would not be present in themonomer and/or fibril.

Antibodies raised to linear peptide regions tend not to be selective foroligomer, and thus bind to monomer as well.

To develop antibodies selective for oligomeric forms of A-beta, theinventors sought to identify regions of sequence in the fibril that areprone to disruption in the context of the fibril, and would be exposedas well on the surface of the oligomer.

As described in the Examples, the inventors identified a regionpredicted to be prone to disruption in the context of the fibril. Theinventors designed cyclic compounds comprising the identified epitope tosatisfy the above criteria of higher curvature, higher exposed surfacearea, and/or alternative dihedral angle distributions.

Antibodies were raised using a cyclic peptide comprising the targetregion that selectively bound the cyclic peptide compared to a linearpeptide of the same sequence (e.g. corresponding linear sequence).Experimental results are described and identify epitope-specific andconformationally selective antibodies that bind synthetic oligomerselectively compared to synthetic monomers, bind CSF from AD patientspreferentially over control CSF and/or bind soluble brain extract fromAD patients preferentially over control soluble brain extract. Furtherstaining of AD brain tissue identified antibodies that show no ornegligible plaque binding and in vitro studies found that the antibodiesinhibited Aβ oligomer propagation and aggregation.

I. Definitions

As used herein, the term ‘A-beta’ may alternately be referred to as‘amyloid beta’, ‘amyloid β’, ‘A-beta’ or ‘Aβ’. Amyloid beta is a peptideof 36-43 amino acids and as used herein includes all wild-type andmutant forms of all species, particularly human A-beta. A-beta40 refersto the 40 amino acid form; A-beta42 refers to the 42 amino acid form,etc. The amino acid sequence of human wildtype A-beta42 is shown in SEQID NO: 31.

As used herein, the term “A-beta monomer” herein refers to an individualsubunit form of A-beta (e.g. 1-40, 1-41, 1-42, 1-43) peptide.

As used herein, the term “A-beta oligomer” refers to a plurality of anyof the A-beta subunits wherein several (e.g. at least two) A-betamonomers are non-covalently aggregated in a conformationally-flexible,partially-ordered, three-dimensional globule of less than about 100, ormore typically less than about 50 monomers. For example, an oligomer maycontain 3 or 4 or 5 or more monomers. The term “A-beta oligomer” as usedherein includes both synthetic A-beta oligomer and/or native A-betaoligomer. “Native A-beta oligomer” refers to A-beta oligomer formed invivo, for example in the brain and CSF of a subject with AD.

As used herein, the term “A-beta fibril” refers to a molecular structurethat comprises assemblies of non-covalently associated, individualA-beta peptides that show fibrillary structure under an electronmicroscope. The fibrillary structure is typically a “cross beta”structure; there is no theoretical upper limit on the size of multimers,and fibrils may comprise thousands or many thousands of monomers.Fibrils can aggregate by the thousands to form senile plaques, one ofthe primary pathological morphologies diagnostic of AD.

The term “alternate conformation than occupied by Q, K, L and/or V inthe linear A-beta peptide, A-beta monomer and/or fibril” or as usedherein means having one or more differing conformational propertiesselected from solvent accessibility, entropy, curvature (e.g. in thecontext of peptide QKLV (SEQ ID NO: 1)), and one or more dihedral anglescompared to said property for Q in linear unstructured A-beta peptide,A-beta monomer and/or A-beta fibril as shown for example in PDB 2MJ4 andshown in FIG. 14. FIG. 4A shows alternate conformational distributionscompared to either the monomer or fibril for residue Q. FIGS. 5 and 6show alternate similar conformational distributions for residues K and Lrespectively and that K and L can be differentiated from both the fibriland monomer. The alternate conformation can be similarly, less or more“constrained” than the comparator conformation. For example FIG. 13demonstrates that Q, K and L in the cyclic compound described are moreconstrained than in the A-beta monomer.

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified L-amino acids. The atoms of the amino acid canfor example include different isotopes. For example, the amino acids cancomprise deuterium substituted for hydrogen nitrogen-15 substituted fornitrogen-14, and carbon-13 substituted for carbon-12 and other similarchanges.

The term “antibody” as used herein is intended to include, monoclonalantibodies, polyclonal antibodies, single chain, veneered, humanized andother chimeric antibodies and binding fragments thereof, including forexample a single chain Fab fragment, Fab′2 fragment or single chain Fvfragment. “Tissue culture supernatant clone” referred to herein refersto monoclonal antibody secreted by a hybridoma into the supernatant forcollection and study. The antibody may be from recombinant sourcesand/or produced in animals such as rabbits, llamas, sharks etc. Alsoincluded are human antibodies that can be produced in transgenic animalsor using biochemical techniques or can be isolated from a library suchas a phage library. Humanized or other chimeric antibodies may includesequences from one or more than one isotype or class or species.

The phrase “isolated antibody” refers to antibody produced in vivo or invitro that has been removed from the source that produced the antibody,for example, an animal, hybridoma or other cell line (such asrecombinant insect, yeast or bacterial cells that produce antibody). Theisolated antibody is optionally “purified”, which means at least: 80%,85%, 90%, 95%, 98% or 99% purity

The term “binding fragment” as used herein to a part or portion of anantibody or antibody chain comprising fewer amino acid residues than anintact or complete antibody or antibody chain and which binds theantigen or competes with intact antibody. Exemplary binding fragmentsinclude without limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv,dimers, nanobodies, minibodies, diabodies, and multimers thereof.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. For example, F(ab′)2 fragments can begenerated by treating the antibody with pepsin. The resulting F(ab′)2fragment can be treated to reduce disulfide bridges to produce Fab′fragments. Papain digestion can lead to the formation of Fab fragments.Fab, Fab′ and F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can also beconstructed by recombinant expression techniques.

The terms “IMGT numbering” or “ImMunoGeneTics database numbering”, whichare recognized in the art, refer to a system of numbering amino acidresidues which are more variable (i.e. hypervariable) than other aminoacid residues in the heavy and light chain variable regions of anantibody, or antigen binding portion thereof.

When an antibody is said to specifically bind to an epitope such as QKLV(SEQ ID NO: 1), what is meant is that the antibody specifically binds toa peptide containing the specified residues or a part thereof forexample at least 2 residues of QKLV with a minimum affinity, and doesnot bind an unrelated sequence or unrelated sequence spatial orientationgreater than for example an isotype control antibody. Such an antibodydoes not necessarily contact each residue of QKLV (SEQ ID NO: 1) andevery single amino acid substitution or deletion within said epitopedoes not necessarily significantly affect and/or equally affect bindingaffinity.

When an antibody is said to selectively bind an epitope such as aconformational epitope, such as QKLV (SEQ ID NO: 1), what is meant isthat the antibody preferentially binds one or more particularconformations containing the specified residues or a part thereof withgreater affinity than it binds said residues in another conformation.For example, when an antibody is said to selectively bind a cyclopeptidecomprising QKLV or related epitope relative to a corresponding linearpeptide, the antibody binds the cyclopeptide with at least a 2 foldgreater affinity than it binds the linear peptide.

As used herein, the term “conformational epitope” refers to an epitopewhere the amino acid sequence has a particular three-dimensionalstructure wherein an aspect of the three-dimensional structure notpresent in a corresponding linear unstructured epitope sequence isrecognized by the cognate antibody. Antibodies which specifically bind aconformation-specific epitope recognize the spatial arrangement of oneor more of the amino acids of that conformation-specific epitope. Forexample a QKLV (SEQ ID NO: 1) conformational epitope, refers to anepitope that is recognized by antibodies selectively, as compared toantibodies raised using linear QKLV (SEQ ID NO: 1). Antibodies whichspecifically and/or selectively bind a conformational epitope recognizethe spatial arrangement of one or more of the amino acids of the epitopesequence specifically and/or selectively. For example a QKLV (SEQ IDNO: 1) conformational epitope, refers to an epitope of QKLV (SEQ IDNO: 1) that is recognized by antibodies specifically and/or selectively,for example at least 2 fold, 3 fold, 5 fold, 10 fold, 50 fold, 100 fold,250 fold, 500 fold or 1000 fold or greater, more selectively as comparedto a corresponding linear QKLV (SEQ ID NO: 1) compound.

The term “constrained conformation” as used herein with respect to anamino acid or a side chain thereof, within a sequence of amino acids, orwith respect to a sequence of amino acids in a larger polypeptide, meansdecreased rotational mobility of the amino acid dihedral angles,relative to a corresponding linear peptide sequence, or the sequence orlarger polypeptide, resulting in a decrease in the number of permissibleconformations. This can be quantified for example by finding the entropyreduction for the ensemble of dihedral angle degrees of freedom, and isplotted in FIG. 13 for QKLV (SEQ ID NO: 1). For example, if the sidechains in the sequence have less conformational freedom than the linearpeptide, the entropy will be reduced. Such conformational restrictionwould enhance the conformational selectivity of antibodies specificallyraised to this antigen. The term “more constrained conformation” as usedherein means that the dihedral angle distribution (ensemble of allowabledihedral angles) of one or more dihedral angles is at least 10% moreconstrained than in the comparator conformation, as determined forexample by the entropy of the amino acids Q, K, L, and/or V.Specifically, the average entropy change relative to the entropy in thefibril, S(constrained)—S(fibril), of QKLV (SEQ ID NO: 1) in the moreconstrained conformational ensemble is on average reduced by more than10% or reduced by more than 20% or reduced by more than 30% or reducedby more than 40%, from the unconstrained conformational ensemble, e.g.of the quantity S(linear)—S(fibril) for the linear peptide (FIG. 13plots this entropy reduction).

The term “related epitope” as used herein means at least two residues ofQKLV (SEQ ID NO: 1), optionally KL, that are antigenic, and/or sequencescomprising up to 1 or up to 2 amino acid residues in a A-beta eitherN-terminal and/or C-terminal to at least two residues of QKLV (SEQ IDNO: 1) (e.g. QKLVF (SEQ ID NO: 10). Exemplary related epitopes caninclude A-beta sequences shown in SEQ ID NO: 1, 2, 9 and 10.

The term “no or negligible plaque binding” or “lacks or has negligibleplaque binding” as used herein with respect to an antibody means thatthe antibody does not show typical plaque morphology staining onimmunohistochemistry (e.g. in situ) and the level of staining iscomparable to or no more than 2 fold the level seen with an IgG negative(e.g. irrelevant) isotype control.

The term “Isolated peptide” refers to peptide that has been produced,for example, by recombinant or synthetic techniques, and removed fromthe source that produced the peptide, such as recombinant cells orresidual peptide synthesis reactants. The isolated peptide is optionally“purified”, which means at least: 80%, 85%, 90%, 95%, 98% or 99% purityand optionally pharmaceutical grade purity.

The term “detectable label” as used herein refers to moieties such aspeptide sequences (such a myc tag, HA-tag, V5-tag or NE-tag),fluorescent proteins that can be appended or introduced into a peptideor compound described herein and which is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the label maybe radio-opaque, positron-emitting radionuclide (for example for use inPET imaging), or a radioisotope, such as ³H, ¹³N, ¹⁴C, ¹⁸F, ³²P, ³⁵S,¹²³I, ¹²⁵I, ¹³¹I; a fluorescent (fluorophore) or chemiluminescent(chromophore) compound, such as fluorescein isothiocyanate, rhodamine orluciferin; an enzyme, such as alkaline phosphatase, beta-galactosidaseor horseradish peroxidase; an imaging agent; or a metal ion. Thedetectable label may be also detectable indirectly for example usingsecondary antibody.

The term “epitope” as commonly used means an antibody binding site,typically a polypeptide segment, in an antigen that is specificallyrecognized by the antibody. As used herein “epitope” can also refer tothe amino acid sequence or a part thereof identified on A-beta using thecollective coordinates method described to which antibodies can beraised using a peptide comprising the epitope sequence. For example anantibody generated against an isolated peptide corresponding to a cycliccompound comprising the identified target region QKLV (SEQ ID NO: 1),recognizes part or all of said “epitope” sequence. An epitope is“accessible” in the context of the present specification when it isaccessible to binding by an antibody.

The term “greater affinity” as used herein refers to a relative degreeof antibody binding where an antibody X binds to target Y more strongly(K_(on)) and/or with a smaller dissociation constant (K_(off)) than totarget Z, and in this context antibody X has a greater affinity fortarget Y than for Z. Likewise, the term “lesser affinity” herein refersto a degree of antibody binding where an antibody X binds to target Yless strongly and/or with a larger dissociation constant than to targetZ, and in this context antibody X has a lesser affinity for target Ythan for Z. The affinity of binding between an antibody and its targetantigen, can be expressed as K_(A) equal to 1/K_(D) where K_(D) is equalto k_(on)/k_(off). The k_(on) and k_(off) values can be measured usingsurface plasmon resonance technology, for example using a MolecularAffinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg,Germany). An antibody that is selective for a conformation presented ina cyclic compound optional a cyclic peptide for example has a greateraffinity for the cyclic compound (e.g. cyclic peptide) compared to acorresponding sequence in linear form (e.g. the sequence non-cyclized).

Also as used herein, the term “immunogenic” refers to substances whichelicit the production of antibodies, activate T-cells and other reactiveimmune cells directed against an antigenic portion of the immunogen.

The term “corresponding linear compound” with regard to a cycliccompound refers to a compound, optionally a peptide, comprising orconsisting of the same sequence or chemical moieties as the cycliccompound but in linear (i.e. non-cyclized) form, for example havingproperties as would be present in solution of a linear peptide. Forexample, the corresponding linear compound can be the synthesizedpeptide that is not cyclized.

As used herein “specifically binds” in reference to an antibody meansthat the antibody recognizes an epitope sequence and binds to its targetantigen with a minimum affinity. For example a multivalent antibodybinds its target with a K_(D) of at least 1e-6, at least 1e-7, at least1e-8, at least 1e-9, or at least 1e-10. Affinities greater than at least1e-8 may be preferred. An antigen binding fragment such as Fab fragmentcomprising one variable domain, may bind its target with a 10 fold or100 fold less affinity than a multivalent interaction with anon-fragmented antibody.

The term “selectively binds” as used herein with respect to an antibodythat selectively binds a form of A-beta (e.g. fibril, monomer oroligomer) or a cyclic compound means that the antibody binds the formwith at least 2 fold, at least 3 fold, or at least 5 fold, at least 10fold, at least 100 fold, at least 250 fold, at least 500 fold or atleast 1000 fold or more greater affinity. Accordingly an antibody thatis more selective for a particular conformation (e.g. oligomer)preferentially binds the particular form of A-beta with at least 2 foldetc., greater affinity compared to another form and/or a linear peptide.

The term “linker” as used herein means a chemical moiety that can becovalently linked to the peptide comprising QKLV (SEQ ID NO: 1) epitopepeptide, optionally linked to QKLV (SEQ ID NO: 1) peptide N- andC-termini to produce a cyclic compound. The linker can comprise a spacerand/or one or more functionalizable moieties. The linker via thefunctionalizable moieties can linked to a carrier protein or animmunogen enhancing agent such as Keyhole Limpet Hemocyanin (KLH).

The term “spacer” as used herein means any non-immunogenic or poorlyimmunogenic chemical moiety that can be covalently-linked directly orindirectly to a peptide N- and C-termini to produce a cyclic compound oflonger length than the peptide itself, for example the spacer can belinked to the N- and C-termini of a peptide consisting of QKLV (SEQ IDNO: 1) to produce a cyclic compound of longer backbone length than theQKLV (SEQ ID NO: 1) sequence itself. That is, the cyclic peptide with aspacer (for example of 3 amino acid residues) makes a larger closedcircle than the cyclic peptide without a spacer. The spacer may include,but are not limited to, non-immunogenic moieties such as G, A, or PEGrepeats, e.g. GQKLV (SEQ ID NO: 4), GQKLVG (SEQ ID NO: 5), GGQKLVG (SEQID NO: 6), GQKLVGG (SEQ ID NO: 7), etc. (see FIG. 12 for specificembodiments). The spacer may comprise or be coupled to one or morefunctionalizing moieties, such as one or more cysteine (C) residues,which can be interspersed within the spacer or covalently linked to oneor both ends of the spacer. Where a functionalizable moiety such as a Cresidue is covalently linked to one or more termini of the spacer, thespacer is indirectly covalently linked to the peptide. The spacer canalso comprise the functionalizable moiety in a spacer residue as in thecase where a biotin molecule is introduced into an amino acid residue.

The term “functionalizable moiety” as used herein refers to a chemicalentity with a “functional group” which as used herein refers to a groupof atoms or a single atom that will react with another group of atoms ora single atom (so called “complementary functional group”) to form achemical interaction between the two groups or atoms. In the case ofcysteine, the functional group can be —SH which can be reacted to form adisulfide bond. Accordingly the linker can for example be CCC. Thereaction with another group of atoms can be covalent or a strongnon-covalent bond, for example as in the case as biotin-streptavidinbonds, which can have Kd˜1e-14. A strong non-covalent bond as usedherein means an interaction with a Kd of at least 1e-9, at least 1e-10,at least 1e-11, at least 1e-12, at least 1e-13 or at least 1e-14.

Proteins and/or other agents may be functionalized (e.g. coupled) to thecyclic peptide, either to aid in immunogenicity, or to act as a probe inin vitro studies. For this purpose, any functionalizable moiety capableof reacting (e.g. making a covalent or non-covalent but strong bond) maybe used. In one specific embodiment, the functionalizable moiety is acysteine residue which is reacted to form a disulfide bond with anunpaired cysteine on a protein of interest, which can be, for example,an immunogenicity enhancing agent such as Keyhole limpet hemocyanin(KLH), or a carrier protein such as Bovine serum albumin (BSA) used forin vitro immunoblots or immunohistochemical assays.

The term “reacts with” as used herein generally means that there is aflow of electrons or a transfer of electrostatic charge resulting in theformation of a chemical interaction.

The term “animal” or “subject” as used herein includes all members ofthe animal kingdom including mammals, optionally including or excludinghumans.

A “conservative amino acid substitution” as used herein, is one in whichone amino acid residue is replaced with another amino acid residuewithout abolishing the protein's desired properties. Suitableconservative amino acid substitutions can be made by substituting aminoacids with similar hydrophobicity, polarity, and R-chain length for oneanother. Examples of conservative amino acid substitution include:

Conservative Substitutions Type of Amino Acid Substitutable Amino AcidsHydrophilic Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr Sulphydryl CysAliphatic Val, Ile, Leu, Met Basic Lys, Arg, His Aromatic Phe, Tyr, Trp

The term “sequence identity” as used herein refers to the percentage ofsequence identity between two polypeptide sequences or two nucleic acidsequences. To determine the percent identity of two amino acid sequencesor of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of afirst amino acid or nucleic acid sequence for optimal alignment with asecond amino acid or nucleic acid sequence). The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % identity=number of identical overlappingpositions/total number of positions.times.100%). In one embodiment, thetwo sequences are the same length. The determination of percent identitybetween two sequences can also be accomplished using a mathematicalalgorithm. A preferred, non-limiting example of a mathematical algorithmutilized for the comparison of two sequences is the algorithm of Karlinand Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modifiedas in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the NBLAST nucleotide programparameters set, e.g., for score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the presentapplication. BLAST protein searches can be performed with the XBLASTprogram parameters set, e.g., to score-50, wordlength=3 to obtain aminoacid sequences homologous to a protein molecule described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. Alternatively, PSI-BLAST can be used to perform aniterated search which detects distant relationships between molecules(Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, thedefault parameters of the respective programs (e.g., of XBLAST andNBLAST) can be used (see, e.g., the NCBI website). Another preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, 1988,CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. The percent identity between twosequences can be determined using techniques similar to those describedabove, with or without allowing gaps. In calculating percent identity,typically only exact matches are counted.

For antibodies, percentage sequence identities can be determined whenantibody sequences maximally aligned by IMGT or other (e.g. Kabatnumbering convention). After alignment, if a subject antibody region(e.g., the entire mature variable region of a heavy or light chain) isbeing compared with the same region of a reference antibody, thepercentage sequence identity between the subject and reference antibodyregions is the number of positions occupied by the same amino acid inboth the subject and reference antibody region divided by the totalnumber of aligned positions of the two regions, with gaps not counted,multiplied by 100 to convert to percentage.

The term “nucleic acid sequence” as used herein refers to a sequence ofnucleoside or nucleotide monomers consisting of naturally occurringbases, sugars and intersugar (backbone) linkages. The term also includesmodified or substituted sequences comprising non-naturally occurringmonomers or portions thereof. The nucleic acid sequences of the presentapplication may be deoxyribonucleic acid sequences (DNA) or ribonucleicacid sequences (RNA) and may include naturally occurring bases includingadenine, guanine, cytosine, thymidine and uracil. The sequences may alsocontain modified bases. Examples of such modified bases include aza anddeaza adenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine. The nucleic acid can be either double stranded or singlestranded, and represents the sense or antisense strand. Further, theterm “nucleic acid” includes the complementary nucleic acid sequences aswell as codon optimized or synonymous codon equivalents. The term“isolated nucleic acid sequences” as used herein refers to a nucleicacid substantially free of cellular material or culture medium whenproduced by recombinant DNA techniques, or chemical precursors, or otherchemicals when chemically synthesized. An isolated nucleic acid is alsosubstantially free of sequences that naturally flank the nucleic acid(i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) fromwhich the nucleic acid is derived.

“Operatively linked” is intended to mean that the nucleic acid is linkedto regulatory sequences in a manner which allows expression of thenucleic acid. Suitable regulatory sequences may be derived from avariety of sources, including bacterial, fungal, viral, mammalian, orinsect genes. Selection of appropriate regulatory sequences is dependenton the host cell chosen and may be readily accomplished by one ofordinary skill in the art. Examples of such regulatory sequencesinclude: a transcriptional promoter and enhancer or RNA polymerasebinding sequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other sequences, such as an origin of replication,additional DNA restriction sites, enhancers, and sequences conferringinducibility of transcription may be incorporated into the expressionvector.

The term “vector” as used herein comprises any intermediary vehicle fora nucleic acid molecule which enables said nucleic acid molecule, forexample, to be introduced into prokaryotic and/or eukaryotic cellsand/or integrated into a genome, and include plasmids, phagemids,bacteriophages or viral vectors such as retroviral based vectors, AdenoAssociated viral vectors and the like. The term “plasmid” as used hereingenerally refers to a construct of extrachromosomal genetic material,usually a circular DNA duplex, which can replicate independently ofchromosomal DNA.

By “at least moderately stringent hybridization conditions” it is meantthat conditions are selected which promote selective hybridizationbetween two complementary nucleic acid molecules in solution.Hybridization may occur to all or a portion of a nucleic acid sequencemolecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,30, 40 or 50) nucleotides in length. Those skilled in the art willrecognize that the stability of a nucleic acid duplex, or hybrids, isdetermined by the Tm, which in sodium containing buffers is a functionof the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(%(G+C)−600/I), or similar equation). Accordingly, theparameters in the wash conditions that determine hybrid stability aresodium ion concentration and temperature. In order to identify moleculesthat are similar, but not identical, to a known nucleic acid molecule a1% mismatch may be assumed to result in about a 1° C. decrease in Tm,for example if nucleic acid molecules are sought that have a >95%identity, the final wash temperature will be reduced by about 5° C.Based on these considerations those skilled in the art will be able toreadily select appropriate hybridization conditions. In preferredembodiments, stringent hybridization conditions are selected. By way ofexample the following conditions may be employed to achieve stringenthybridization: hybridization at 5× sodium chloride/sodium citrate(SSC)/5×Denhardt's solution/1.0% SDS at Tm−5° C. based on the aboveequation, followed by a wash of 0.2× SSC/0.1% SDS at 60° C. Moderatelystringent hybridization conditions include a washing step in 3× SSC at42° C. It is understood, however, that equivalent stringencies may beachieved using alternative buffers, salts and temperatures. Additionalguidance regarding hybridization conditions may be found in: CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold SpringHarbor Laboratory Press, 2001.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment. For example, a subject with early stageAD can be treated to prevent progression can be treated with a compound,antibody, immunogen, nucleic acid or composition described herein toprevent progression.

The term “administered” as used herein means administration of atherapeutically effective dose of a compound or composition of thedisclosure to a cell or subject.

As used herein, the phrase “effective amount” means an amount effective,at dosages and for periods of time necessary to achieve a desiredresult. Effective amounts when administered to a subject may varyaccording to factors such as the disease state, age, sex, weight of thesubject. Dosage regime may be adjusted to provide the optimumtherapeutic response.

The term “pharmaceutically acceptable” means that the carrier, diluent,or excipient is compatible with the other components of the formulationand not substantially deleterious to the recipient thereof.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

In understanding the scope of the present disclosure, the term“consisting” and its derivatives, as used herein, are intended to beclose ended terms that specify the presence of stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The recitation of numerical ranges by endpoints herein includes allnumbers and fractions subsumed within that range (e.g. 1 to 5 includes1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood thatall numbers and fractions thereof are presumed to be modified by theterm “about.” Further, it is to be understood that “a,” “an,” and theinclude plural referents unless the content clearly dictates otherwise.The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%,preferably 10-20%, more preferably 10% or 15%, of the number to whichreference is being made.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects of the invention are defined in more detail. Each aspect sodefined may be combined with any other aspect or aspects unless clearlyindicated to the contrary. In particular, any feature indicated as beingpreferred or advantageous may be combined with any other feature orfeatures indicated as being preferred or advantageous.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

II. Epitopes and Binding Proteins

The inventors have identified an epitope in A-beta, QKLV (SEQ ID NO: 1)at amino acids 15 to 18 on A-beta. They have further identified that theepitope or a part thereof may be a conformational epitope, and that QKLV(SEQ ID NO: 1) may be selectively accessible to antibody binding inoligomeric species of A-beta.

Without wishing to be bound by theory, fibrils may present interactionsites that have a propensity to catalyze oligomerization. This may bestrain-specific, and may only occur when selective fibril surface notpresent in normal patients is exposed and thus able to have aberrantinteractions with the monomer (i.e. is presented to the monomer).Environmental challenges such as low pH, osmolytes present duringinflammation, or oxidative damage may induce disruption in fibrils thatcan lead to exposure of more weakly stable regions. There is ofinterest, then, to predict these weakly-stable regions, and use suchpredictions to rationally design antibodies that could target them.Regions likely to be disrupted in the fibril may also be good candidatesfor exposed regions in oligomeric species.

Computer based systems and methods to predict contiguous protein regionsthat are prone to disorder are described in U.S. Patent Application Ser.No. 62/253,044, SYSTEMS AND METHODS FOR PREDICTING MISFOLDED PROTEINEPITOPES BY COLLECTIVE COORDINATE BIASING filed Nov. 9, 2015 and U.S.patent application Ser. No. 12/574,637, “Methods and Systems forPredicting Misfolded Protein Epitopes” filed Oct. 6, 2009, each of whichare hereby incorporated by reference in their entirety. As described inthe Examples, the methods were applied to A-beta and identified anepitope that may be more accessible in A-beta oligomers.

As described in the Examples, a cyclic peptide cyclo(CGQKLVG) (SEQ IDNO: 3) may capture the conformational differences of the epitope inoligomers relative to the monomer and/or fibril species. For example,differences in solvent accessible surface, curvature and the dihedralangle distributions for several of the dihedral angles in the cyclic7-mer cyclo (CGQKLVG) (SEQ ID NO: 3) were found to be substantiallydifferent than in the A-beta monomer and fibril, suggesting that thecyclopeptide provides for a conformational epitope that is distinct fromthe linear unstructured epitope.

Antibodies raised using an immunogen comprising cyclo(CGQKLVG) (SEQ IDNO: 2) selectively bound cyclo(CGQKLVG) (SEQ ID NO: 2) over linearCGQKLVG (SEQ ID NO: 2) and selectively bound synthetic and/or nativeoligomeric A-beta species compared to monomeric A-beta and A-beta fibrilplaques. Further, antibodies raised to cyclo(CGQKLVG) (SEQ ID NO: 2)were able to inhibit in vitro propagation of A-beta aggregation andinhibit A-beta oligomer induced toxicity in a neural cell model.

i) QKLV (SEQ ID NO: 1) “Epitope” Compounds

Accordingly, the present disclosure identifies a conformational epitopein A-beta consisting of amino acids QKLV (SEQ ID NO: 1), or a partthereof, which correspond to amino acids residues 15-18 on A-beta.

An aspect includes a compound comprising an A-beta peptide comprising orconsisting of QKLV (SEQ ID NO: 1) or sequence of a related epitopeand/or part of any of the foregoing.

In some embodiments, the A-beta peptide comprising QKLV (SEQ ID NO: 1)can include 1, 2 or 3 additional residues in A-beta N- and/or C-terminusof QKLV (SEQ ID NO: 1) for example HQKLV (SEQ ID NO: 2). The 3 aminoacids N-terminal to QKLV (SEQ ID NO: 1) in A-beta are VHH and the 3amino acids C-terminal to QKLV (SEQ ID NO: 1) are FFA. In an embodimentthe A-beta peptide is a maximum of 7 amino acids, 6 amino acids or 5amino acids.

In an embodiment, the A-beta peptide comprises one or two additionalresidues in A-beta that are C-terminal to QKLV (SEQ ID NO: 1).

In an embodiment, the compound comprises a sequence listed in Table 10.

In an embodiment, the compound further includes a linker. The linkercomprises a spacer and/or one or more functionalizable moieties. Thelinker can for example comprise 3, 4, 5, 6, 7 or 8 amino acids and/orequivalently functioning molecules such as polyethylene glycol (PEG)moieties, and/or a combination thereof. In an embodiment, the spaceramino acids are selected from non-immunogenic or poorly immunogenicamino acid residues such as G and A, for example the spacer can be GGG,GAG, G(PEG)G, PEG-PEG-GG and the like. One or more functionalizablemoieties e.g. amino acids with a functional group may be included forexample for coupling the compound to an agent or detectable tag or acarrier such as BSA or an immunogenicity enhancing agent such as KLH.

In an embodiment the linker comprises GC-PEG, PEG-GC, GCG or PEG-C-PEG.

In an embodiment, the linker comprises 1, 2, 3, 4, 5, 6, 7 or 8 aminoacids.

In embodiments wherein the A-beta peptide comprising QKLV (SEQ ID NO: 1)includes 1, 2 or 3 additional residues found in A-beta that are N-and/or C-terminal to QKLV (SEQ ID NO: 1) (e.g. HQKLV (SEQ ID NO: 2), thelinker in the cyclized compound is covalently linked to the N- and/orC-termini of the A-beta residues (e.g. to residues H and V). The cycliccompound can be synthesized as a linear molecule with the linkercovalently attached to the N-terminus or C-terminus of the peptidecomprising QKLV (SEQ ID NO: 1) prior to cyclization. Alternatively partof the linker is covalently attached to the N-terminus and part iscovalently attached to the C-terminus prior to cyclization. In eithercase, the linear compound is cyclized for example in a head to tailcyclization (e.g. amide bond cyclization).

Proteinaceous portions of compounds may be prepared by chemicalsynthesis using techniques well known in the chemistry of proteins suchas solid phase synthesis or synthesis in homogenous solution.

In an embodiment, the compound is a cyclic compound. In an embodiment,the cyclic compound is a cyclic peptide (cyclopeptide).

Reference to the “cyclic peptide” herein can refer to a fullyproteinaceous compound (e.g. wherein the linker is for example 1, 2, 3,4, 5, 6, 7 or 8 amino acids). It is understood that properties describedfor the cyclic peptide determined in the examples can be incorporated inother compounds (e.g. cyclic compounds) comprising non-amino acid linkermolecules.

An aspect therefore provides a cyclic compound comprising peptide QKLV(SEQ ID NO: 1) (e.g. A beta peptide) and a linker, wherein the linker iscovalently coupled to the peptide comprising QKLV (SEQ ID NO: 1)(optionally the Q and the V residues when the peptide consists of QKLV(SEQ ID NO: 1)), optionally wherein at least Q is in an alternateconformation than Q in a linear peptide comprising QKLV (SEQ ID NO: 1),and/or the conformation of Q in the monomer and/or fibril, andoptionally wherein at least Q, or at least K, or at least L is in a moreconstrained conformation in the cyclic compound than the conformationoccupied in a linear peptide comprising QKLV (SEQ ID NO: 1) or a relatedepitope sequence.

The linear peptide comprising the A-beta sequence can be comprised in alinear compound. The linear compound or the linear peptide comprisingQKLV (SEQ ID NO: 1) is in an embodiment, a corresponding linear peptide.In another embodiment, the linear peptide is any length of A-betapeptide comprising QKLV (SEQ ID NO: 1), including for example a linearpeptide comprising A-beta residues 1-35, or smaller portions thereofsuch as A-beta residues 10-20, 11-20, 12-20, 13-20, 10-19, 10-18 and thelike etc. The linear peptide can in some embodiments also be a fulllength A-beta peptide.

In an embodiment the cyclic compound comprises peptide comprising orconsisting of QKLV (SEQ ID NO: 1) and a linker, wherein the linker iscoupled to the N- and C-termini of the peptide (e.g. the Q and the Vresidues when the peptide consists of QKLV (SEQ ID NO: 1)). In anembodiment, at least Q is in an alternate conformation in the cycliccompound than occupied by Q in a linear peptide comprising QKLV (SEQ IDNO: 1). In an embodiment, at least Q is in an alternate conformation inthe cyclic compound than occupied by Q in the monomer and/or fibril.

In an embodiment, the alternate conformation is a constrainedconformation.

In an embodiment, at least Q, optionally alone or in combination with atleast K, or at least L is in a more constrained conformation than theconformation occupied in a linear peptide comprising QKLV (SEQ ID NO:1).

In an embodiment, the conformation of Q and/or Q in combination with oneor more of K, L and/or V is comprised in the compound in an alternateconformation than that occupied in the linear peptide comprising QKLV,optionally in a more constrained conformation.

For example, the alternate conformation can include one or morediffering dihedral angles in residues Q, and optionally in Q and/or K,and/or L and/or V differing from the dihedral angles in the linearpeptide or peptide in the context of the fibril.

The alternate conformation can include for one or more amino acid sidechains of the epitope, particularly for Q and also for K or L, anincrease or decrease in the solvent accessible surface area (SASA) ofone or more parts of the side chains relative to a linear peptide and/orA-beta fibril.

For example, FIG. 10 (top panel) and FIG. 11 show that the side chainsof Q and L are more solvent-exposed in the cyclic compound than in thelinear peptide due to the bowing of the backbone, which “splays out” theside chains. FIG. 11, which looks at the specific moieties on the sidechains, demonstrates that the increase in SASA of Q comes from theC_(γ)H₂ group and/or the N_(ε)H₂ groups. Similarly it can be seen thatthe increase in SASA of L comes from C_(δ1)H₃ and/or C_(δ2)H₃.

In an embodiment, the SASA of Q, and optionally one or more of K or L inthe compound is increased relative to the linear peptide QKLV (SEQ IDNO: 1).

The alternate conformation can also include an increase in curvaturecentered around an amino acid or of the cyclic peptide QKLV (SEQ IDNO: 1) relative to a linear peptide and/or A-beta fibril.

In an embodiment, the alternate conformation QKLV (SEQ ID NO: 1) has anincreased curvature relative to linear QKLV (SEQ ID NO: 1). As shown inthe Examples, the curvature of the cyclic epitope backbone is alsoincreased relative to that in the linear peptide or peptide in thecontext of the fibril (FIG. 3) as described in Example 4.

The values of the curvature were determined for Q, K, L, V incyclo(CGQKLVG) (SEQ ID NO: 3), linear CGQKLVG (SEQ ID NO: 3), and QKLV(SEQ ID NO: 1) in the context of the fibril. As described in Example 4,these were:

-   Cyclic peptide: 1.248 1.566 1.422 1.46-   Linear Peptide: 0.870 1.355 0.931 1.303-   Fibril: 0.740 1.159 0.796 1.188-   The averages of these are:-   Cyclic peptide: 1.42-   Linear peptide: 1.11-   Fibril: 0.97

Accordingly, the curvature of the Q, and/or one or more of K, L and/or Vin the alternate conformation is increased by at least 0.1, 0.2, 0.3 ormore radians compared to the linear peptide.

In an embodiment, the QK, QKL and/or QKLV (SEQ ID NO: 1) are in analternate conformation, for example as compared to what is occupied bythese residues in a non-oligomeric conformation.

For example for Q and K, the SASA weighted by the solubility isincreased in the cyclic peptide relative to the linear peptide orpeptide in fibril.

Further the entropy of the side chains is reduced in the cyclic peptiderelative to the linear peptide, rendering the side chains in a morestructured conformation than the linear peptide.

Conformations preferred in the cyclic compound are different from theconformations preferred in either the linear peptide or fibril.Specifically, the dihedral angle between the Cα-Cβ-Cγ-Cδ atoms in theside chain of Q is different between the cyclic peptide and the linearpeptide. This is depicted in FIG. 14, as well as in FIGS. 4, 5, and 6.

In FIG. 14, the solid arrow indicates the dihedral angle typicallyoccupied in the cyclic peptide, and the dashed arrow indicates thedihedral angle typically occupied in the linear peptide, with thatportion of the side chain shown as semi-transparent.

As demonstrated herein, the curvature of the cyclic epitope is increasedrelative that in the linear peptide or to the peptide in the context ofthe fibril (FIG. 3). It is also demonstrated, that one or more of thedihedral angles in residues Q, and/or K, and/or L and/or V aresignificantly different from the dihedral angles in the linear peptideor peptide in the context of the fibril. Further, for one or more aminoacids of the epitope, particularly Q and K, the solvent accessiblesurface area (SASA) of side chains are increased in the cyclic peptideas compared to the peptide in the context of the fibril. For these aminoacids, the SASA weighted by the solubility is increased in the cyclicpeptide relative to the peptide in fibril. In addition, the entropy ofthe side chains is reduced in the cyclic peptide relative to the linearpeptide, rendering the side chains in a more structured conformationthan the linear peptide (FIG. 13).

Cyclic compounds which show similar changes are also encompassed.

The cyclic compound in some embodiments that comprises a peptidecomprising QKLV (SEQ ID NO: 1) can include 1, 2, 3 or more residues inA-beta upstream and/or downstream of QKLV (SEQ ID NO: 1). In such casesthe spacer is covalently linked to the N- and C-termini of the A-betaresidues.

In an embodiment, the cyclic compound is a compound in FIG. 12.

Methods for making cyclized peptides are known in the art and includeSS-cyclization or amide cyclization (head-to-tail, or backbonecyclization). Methods are further described in Example 5. For example, apeptide with “C” residues at its N- and C-termini, e.g. CGQKLVGC (SEQ IDNO: 8), can be reacted by SS-cyclization to produce a cyclic peptide.

The cyclic compound can be synthesized as a linear molecule with thelinker covalently attached to the N-terminus or C-terminus of thepeptide comprising the A-beta peptide, prior to cyclization.Alternatively part of the linker is covalently attached to theN-terminus and part is covalently attached to the C-terminus prior tocyclization. In either case, the linear compound is cyclized for examplein a head to tail cyclization (e.g. amide bond cyclization).

In some embodiments, the linker or spacer is indirectly coupled to theN- and C-terminus residues of the A-beta peptide.

As described in Example 4, the cyclic compound of FIG. 12A was assessedfor its relatedness to the conformational epitope identified. The cycliccompound comprising QKLV (SEQ ID NO: 1) peptide for example can be usedto raise antibodies selective for one or more conformations.

The epitope QKLV (SEQ ID NO: 1) and/or a part thereof, as describedherein may be a potential target in misfolded propagating strains ofA-beta involved in A-beta, and antibodies that recognize theconformational epitope may for example be useful in detecting suchpropagating strains.

Also provided in another aspect is an isolated peptide comprising anA-beta peptide sequence described herein, including linear peptides andcyclic peptides. Linear peptides can for example be used for selectingantibodies for lack of binding thereto. The isolated peptide cancomprise a linker sequence described herein. The linker can becovalently coupled to the N or C terminus or may be partially coupled tothe N terminus and partially coupled to the C terminus as in CGQKLVG(SEQ ID NO: 3) linear peptide. In the cyclic peptide, the linker iscoupled to the C-terminus and N-terminus directly or indirectly.

Another aspect includes an immunogen comprising a compound, optionally acyclic compound described herein (e.g. comprising for example QKL, KLVor QKLV (SEQ ID NO: 1).

An immunogen is suitably prepared or formulated for administration to asubject, for example, the immunogen may be sterile, or purified. In anembodiment, the immunogen is a cyclic peptide comprising QKLV (SEQ IDNO: 1) or a related epitope sequence.

In an embodiment, the immunogen comprises immunogenicity enhancing agentsuch as Keyhole Limpet Hemocyanin (KLH). The immunogenicity enhancingagent can be coupled to the compound either directly, such as through anamide bound, or indirectly through a functionalizable moiety in thelinker. When the linker is a single amino acid residue (for example withthe A-beta peptide in the cyclic compound is 6 amino acid residues) thelinker can be the functionalizable moiety (e.g. a cysteine residue).

The immunogen can be produced by conjugating the cyclic compoundcontaining the constrained epitope peptide to an immunogenicityenhancing agent such as Keyhole Limpet Hemocyanin (KLH) or a carriersuch bovine serum albumin (BSA) using for example the method describedin Lateef et al 2007, herein incorporated by reference. In anembodiment, the method described in Example 3 or 4 is used.

A further aspect is an isolated nucleic acid encoding the proteinaceousportion of a compound or immunogen described herein. In embodiment, thenucleic acid molecule encodes any one of the amino acid sequences sentforth in SEQ ID NOs: 1-10.

In an embodiment, nucleic acid molecule encodes QKLV (SEQ ID NO: 1) or arelated epitope and optionally a linker described herein.

A further aspect is a vector comprising said nucleic acid. Suitablevectors are described elsewhere herein.

ii) Antibodies, Cells and Nucleic Acids

The compounds and particularly the cyclic compounds described above canbe used to raise antibodies that specifically bind QKLV (SEQ ID NO: 1)in A-beta and/or which recognize specific conformations of QKLV (SEQ IDNO: 1) in A-beta. As demonstrated in the Examples, the cyclic compoundCGQKLVG (SEQ ID NO: 3) was immunogenic, and produced a number ofantibodies that specifically and/or selectively bind the cyclic compoundrelative to the corresponding linear peptide. In addition, antibodiesraised to CGQKLVG (SEQ ID NO: 3) specifically and/or selectively boundA-beta oligomers, lacked or had negligible plaque binding, inhibitedpropagation of A-beta aggregation and inhibited A-beta oligomer inducedneural toxicity in vitro.

Accordingly, the compounds and particularly the cyclic compoundsdescribed above can be used to raise antibodies that specifically bindQKLV (SEQ ID NO: 1) in A-beta and/or which recognize specificconformations of these residues in A-beta, including one or moredifferential features described herein. Similarly cyclic compoundscomprising for example related epitope sequences described herein suchas SEQ ID NO: 1, 2, 9 or 10, can be used to raise antibodies thatspecifically selectively bind conformational epitopes of QKLV (SEQ IDNO: 1).

Accordingly, an aspect includes an antibody (including a bindingfragment thereof) that specifically binds to an A-beta peptide having asequence QKLV (SEQ ID NO: 1) or a related epitope sequence, for exampleas set forth in any one of SEQ ID NO: 1, 2, 9 or 10.

In an embodiment, the A-beta peptide is comprised in a cyclic compound,optionally a cyclic peptide, and the antibody is specific or selectivefor the portion of A-beta presented in the cyclic compound.

In an embodiment, the antibody selectively binds A-beta peptide in acyclic compound, in the context of cyclo (CGQKLVG) (SEQ ID NO: 3)relative to a linear peptide comprising QKLV (SEQ ID NO: 1), optionallyin the context of linear CGQKLVG (SEQ ID NO: 3), e.g. correspondinglinear sequence. For example, in an embodiment the antibody selectivelybinds QKLV (SEQ ID NO: 1) in a cyclic conformation and has at least 2fold, at least 3 fold, at least 5 fold, at least 10 fold at least 20fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100fold, at least 500 fold, at least 1000 fold more selective greaterselectivity QKLV (SEQ ID NO: 1) in the cyclic conformation compared toQKLV (SEQ ID NO: 1) in a linear unstructured compound, for example asmeasured by ELISA or surface plasmon resonance, or optionally using amethod described herein.

In an embodiment, the antibody specially and/or selectively binds theA-beta peptide of the cyclic compound, wherein the A-beta has a sequenceas set forth in any one of SEQ ID NOs: 1, 2, 4 to 10.

In an embodiment, the antibody selectively binds to an A-beta portion ofa sequence of any of SEQ ID NOs: 1-10 presented in a cyclic compound,compared to a linear compound.

In an embodiment, the cyclic compound is a cyclic peptide. In anembodiment, A-beta peptide in the cyclic peptide is any one of SEQ IDNO: 1, 2, 9 or 10. In an embodiment, the cyclic peptide comprises anyone of the sequences in SEQ ID NO: 1-10.

As described in the examples, antibodies having one or more propertiescan be selected using assays described in the Examples.

In an embodiment, the antibody does not bind a linear peptide comprisingthe sequence QKLV (SEQ ID NO: 1), optionally wherein the sequence of thelinear peptide is a linear version of a cyclic sequence used to raisethe antibody as described herein, optionally as set forth in SEQ ID NO:3.

In an embodiment, the antibody is a conformation specific and/orselective A-beta antibody. For example an antibody that binds aparticular epitope conformation can be referred to as a conformationspecific antibody. Such antibodies can be selected using the methodsdescribed herein. The conformation specific antibody can differentiallyrecognize a particular A-beta species or a group of related species(e.g. dimers, trimers, and other oligomeric species) and can have ahigher affinity for one species or group of species compared to another(e.g. to either the A-beta monomer or fibril species).

In an embodiment the antibody is isolated.

In an embodiment, the antibody is an exogenous antibody.

In an embodiment, the antibody specifically binds an epitope on A-beta,the epitope comprising or consisting of QKLV (SEQ ID NO: 1), a relatedepitope or a part thereof.

As described in the Examples, Q and/or QK and/or QL residues may bepredominantly accessible or exposed in conformations of A-beta that aredistinct from the monomer and/or fibril forms.

Accordingly a further aspect is an antibody which specifically binds anepitope on A-beta, wherein the epitope comprises or consists of at leastone amino acid residue predominantly involved in binding to theantibody, wherein the at least one amino acid is Q, K or L embeddedwithin the sequence QKLV (SEQ ID NO: 1).

In an embodiment, the epitope comprises or consists of at least twoconsecutive amino acid residues predominantly involved in binding to theantibody, wherein the at least two consecutive amino acids are QK or KLembedded within QKLV (SEQ ID NO: 1).

In another embodiment, the epitope consists of QKLV (SEQ ID NO: 1).

In an embodiment, the antibody does not specifically bind monomericA-beta. In an embodiment, the antibody does not specifically bind A-betasenile plaques, for example in situ in AD brain tissue.

In another embodiment, the antibody does not selectively bind monomericA-beta compared to native- or synthetic-oligomeric A-beta.

In an embodiment, the antibody specifically and/or selectively binds aspecies of A-beta selectively such as A-beta oligomer. In an embodiment,the selectivity is at least 3 fold, at least 5 fold, at least 10 fold,at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold,at least 100 fold, at least 500 fold, at least 1000 fold more selectivefor A-beta oligomer over a species of A-beta selected from A-betamonomer and/or A-beta fibril and/or a linear compound comprising QKLV(SEQ ID NO: 1).

In an embodiment, the A-beta oligomer comprises A-beta 1-42 subunits.

In an embodiment, the antibody lacks A-beta fibril plaque (also referredto as senile plaque) staining. Absence of plaque staining can beassessed by comparing to a positive control such as A-beta-specificantibodies 6E10 and 4G8 (Biolegend, San Diego, Calif.), or 2C8 (EnzoLife Sciences Inc., Farmingdale, N.Y.), or any other antibody reactiveto fibrillar forms of A-beta, and an isotype control. An antibodydescribed herein lacks or has negligible A-beta fibril plaque stainingif the antibody does not show typical plaque morphology staining and thelevel of staining is comparable to or no more than 2 fold the level seenwith an IgG negative isotype control. The scale can for example set thelevel of staining with isotype control at 1 and with 6E10 at 10. Anantibody lacks A-beta fibril plaque staining if the level of staining onsuch a scale is 2 or less. In embodiment, the antibody shows minimalA-beta fibril plaque staining, for example on the foregoing scale,levels scored at less about or less than 3.

In an embodiment, the antibody is a monoclonal antibody.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from a subject immunized with an described herein, andfused with myeloma cells by standard somatic cell fusion procedures thusimmortalizing these cells and yielding hybridoma cells. Such techniquesare well known in the art, (e.g. the hybridoma technique originallydeveloped by Kohler and Milstein (Nature 256:495-497 (1975)) as well asother techniques such as the human B-cell hybridoma technique (Kozbor etal., Immunol. Today 4:72 (1983)), the EBV-hybridoma technique to producehuman monoclonal antibodies (Cole et al., Methods Enzymol, 121: 140-67(1986)), and screening of combinatorial antibody libraries (Huse et al.,Science 246:1275 (1989)). Hybridoma cells can be screenedimmunochemically for production of antibodies specifically reactive withthe amyotrophic lateral sclerosis-specific epitopes and the monoclonalantibodies can be isolated.

Specific antibodies, or antibody fragments, reactive against particularantigens or molecules, may also be generated by screening expressionlibraries encoding immunoglobulin genes, or portions thereof, expressedin bacteria with cell surface components. For example, complete Fabfragments, VH regions and FV regions can be expressed in bacteria usingphage expression libraries (see for example Ward et al., Nature41:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); andMcCafferty et al., Nature 348:552-554 (1990).

In an embodiment, the antibody is a humanized antibody.

The humanization of antibodies from non-human species has been welldescribed in the literature. See for example EP-B1 0 239400 and Carter &Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997 incorporated byreference in their entirety herein). Humanized antibodies are alsoreadily obtained commercially (eg. Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.).

Humanized forms of rodent antibodies are readily generated by CDRgrafting (Riechmann et al. Nature, 332:323-327, 1988). In this approachthe six CDR loops comprising the antigen binding site of the rodentmonoclonal antibody are linked to corresponding human framework regions.CDR grafting often yields antibodies with reduced affinity as the aminoacids of the framework regions may influence antigen recognition (Foote& Winter. J Mol Biol, 224: 487-499, 1992). To maintain the affinity ofthe antibody, it is often necessary to replace certain frameworkresidues by site directed mutagenesis or other recombinant techniquesand may be aided by computer modeling of the antigen binding site (Co etal. J Immunol, 152: 2968-2976, 1994).

Humanized forms of antibodies are optionally obtained by resurfacing(Pedersen et al. J Mol Biol, 235: 959-973, 1994). In this approach onlythe surface residues of a rodent antibody are humanized.

Human antibodies specific to a particular antigen may be identified by aphage display strategy (Jespers et al. Bio/Technology, 12: 899-903,1994). In one approach, the heavy chain of a rodent antibody directedagainst a specific antigen is cloned and paired with a repertoire ofhuman light chains for display as Fab fragments on filamentous phage.The phage is selected by binding to antigen. The selected human lightchain is subsequently paired with a repertoire of human heavy chains fordisplay on phage, and the phage is again selected by binding to antigen.The result is a human antibody Fab fragment specific to a particularantigen. In another approach, libraries of phage are produced weremembers display different human antibody fragments (Fab or Fv) on theirouter surfaces (Dower et al., WO 91/17271 and McCafferty et al., WO92/01047). Phage displaying antibodies with a desired specificity areselected by affinity enrichment to a specific antigen. The human Fab orFv fragment identified from either approach may be recloned forexpression as a human antibody in mammalian cells.

Human antibodies are optionally obtained from transgenic animals (U.S.Pat. Nos. 6,150,584; 6,114,598; and 5,770,429). In this approach theheavy chain joining region (JH) gene in a chimeric or germ-line mutantmouse is deleted. Human germ-line immunoglobulin gene array issubsequently transferred to such mutant mice. The resulting transgenicmouse is then capable of generating a full repertoire of humanantibodies upon antigen challenge.

Antibodies, including humanized or human antibodies, are selected fromany class of immunoglobulins including: IgM, IgG, IgD, IgA or IgE; andany isotype, including: IgG1, IgG2, IgG3 and IgG4. Chimeric, humanizedor human antibodies may include sequences from one or more than oneisotype or class.

Further, these antibodies are typically produced as antigen bindingfragments such as Fab, Fab′ F(ab′)2, Fd, Fv and single domain antibodyfragments, or as single chain antibodies in which the heavy and lightchains are linked by a spacer. Also, the human or humanized antibodiesmay exist in monomeric or polymeric form. The humanized antibodyoptionally comprises one non-human chain and one humanized chain (i.e.one humanized heavy or light chain).

Additionally, antibodies specific for the epitopes described herein arereadily isolated by screening antibody phage display libraries. Forexample, an antibody phage library is optionally screened by using adisease specific epitope of the current invention to identify antibodyfragments specific for the disease specific epitope. Antibody fragmentsidentified are optionally used to produce a variety of recombinantantibodies that are useful with different embodiments of the presentinvention. Antibody phage display libraries are commercially available,for example, through Xoma (Berkeley, Calif.) Methods for screeningantibody phage libraries are well known in the art.

A further aspect is antibody and/or binding fragment thereof comprisinga light chain variable region and a heavy chain variable region, theheavy chain variable region comprising complementarity determiningregions CDR-H1, CDR-H2 and CDR-H3, the light chain variable regioncomprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3and with the amino acid sequences of said CDRs comprising the sequencesset forth below:

Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GYTFTDYE 11 CDR-H2 IDPETGDT 12CDR-H3 TSPIYYDYDWFAY 13 Light CDR-L1 QSLLNNRTRKNY 14 CDR-L2 WAS 15CDR-L3 KQSYNLRT 16 and/or Chain CDR Sequence SEQ ID NO Heavy CDR-H1GFSLSTSGMG 21 CDR-H2 IWWDDDK 22 CDR-H3 ARSITTVVATPFDY 23 Light CDR-L1QNVRSA 24 CDR-L2 LAS 25 CDR-L3 LQHWNSPFT 26

In an embodiment, the antibody is a monoclonal antibody. In anembodiment, the antibody is a chimeric antibody such as a humanizedantibody comprising the CDR sequences as recited in Tables 6 and/or 8.

Also provided in another embodiment, is an antibody comprising the CDRsin Table 6 and/or 8 and a light chain variable region and a heavy chainvariable region, optionally in the context of a single chain antibody.

In yet another aspect, the antibody comprises a heavy chain variableregion comprises: i) an amino acid sequence as set forth in SEQ ID NO:18 or 28; ii) an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80% sequence identity to SEQ ID NO: 18 or 28,wherein the CDR sequences are as set forth in SEQ ID NO: 11, 12, 13, 21,22, and/or 23, or iii) a conservatively substituted amino acid sequencei). In another aspect the antibody comprises a light chain variableregion comprising i) an amino acid sequence as set forth in SEQ ID NO:20 or 30, ii) an amino acid sequence with at least 50%, at least 60%, atleast 70%, at least 80% 70% sequence identity to SEQ ID NO: 20 or 30,wherein the CDR sequences are as set forth in SEQ ID NO: 14, 15, 16, 24,25 an/or 26, or iii) a conservatively substituted amino acid sequence ofi). In another embodiment, the heavy chain variable region amino acidsequence is encoded by a nucleotide sequence as set out in SEQ ID NO: 17or 27 or a codon degenerate optimized version thereof. In anotherembodiment, the antibody comprises a light chain variable region aminoacid sequence encoded by a nucleotide sequence as set out in SEQ ID NO:19 or 29 or a codon degenerate or optimized version thereof. In anembodiment, the heavy chain variable region comprises an amino acidsequence as set forth in SEQ ID NO: 18 or 28. In an embodiment, thelight chain variable region comprises an amino acid sequence as setforth in SEQ ID NO: 20 or 30.

Another aspect is an antibody that specifically binds a same epitope asthe antibody with CDR sequences as recited in Table 6 and/or 8.

Another aspect includes an antibody that competes for binding to humanA-beta with an antibody comprising the CDR sequences as recited in Table6 and/or 8.

Competition between antibodies can be determined for example using anassay in which an antibody under test is assessed for its ability toinhibit specific binding of a reference antibody to the common antigen.A test antibody competes with a reference antibody if an excess of atest antibody (e.g., at least a 2 fold, 5, fold, 10 fold or 20 fold)inhibits binding of the reference antibody by at least 50%, at least75%, at least 80%, at least 90% or at least 95% as measured in acompetitive binding assay.

A further aspect is an antibody conjugated to a therapeutic, detectablelabel or cytotoxic agent. In an embodiment, the detectable label is apositron-emitting radionuclide. A positron-emitting radionuclide can beused for example in PET imaging.

A further aspect relates to an antibody complex comprising an antibodydescribed herein and/or a binding fragment thereof and oligomericA-beta.

A further aspect is an isolated nucleic acid encoding an antibody orpart thereof described herein.

Nucleic acids encoding a heavy chain or a light chain are also provided,for example encoding a heavy chain comprising CDR-H1, CDR-H2 and/orCDR-H3 regions described herein or encoding a light chain comprisingCDR-L1, CDR-L2 and/or CDR-L3 regions described herein.

The present disclosure also provides variants of the nucleic acidsequences that encode for the antibody and/or binding fragment thereofdisclosed herein. For example, the variants include nucleotide sequencesthat hybridize to the nucleic acid sequences encoding the antibodyand/or binding fragment thereof disclosed herein under at leastmoderately stringent hybridization conditions or codon degenerate oroptimized sequences In another embodiment, the variant nucleic acidsequences have at least 50%, at least 60%, at least 70%, most preferablyat least 80%, even more preferably at least 90% and even most preferablyat least 95% sequence identity to nucleic acid sequences encoding aproteinaceous sequence herein including any one of SEQ ID NOs: 18, 20,28 and 30.

In an embodiment, the nucleic acid is an isolated nucleic acid.

Another aspect is an expression cassette or a vector comprising thenucleic acid herein disclosed. In an embodiment, the vector is anisolated vector.

The vector can be any vector, including vectors suitable for producingan antibody and/or binding fragment thereof or expressing an epitopepeptide sequence described herein.

The nucleic acid molecules may be incorporated in a known manner into anappropriate expression vector which ensures expression of the protein.Possible expression vectors include but are not limited to cosmids,plasmids, or modified viruses (e.g. replication defective retroviruses,adenoviruses and adeno-associated viruses). The vector should becompatible with the host cell used. The expression vectors are “suitablefor transformation of a host cell”, which means that the expressionvectors contain a nucleic acid molecule encoding the peptidescorresponding to epitopes or antibodies described herein.

In an embodiment, the vector is suitable for expressing for examplesingle chain antibodies by gene therapy. The vector can be adapted forspecific expression in neural tissue, for example using neural specificpromoters and the like. In an embodiment, the vector comprises an IRESand allows for expression of a light chain variable region and a heavychain variable region. Such vectors can be used to deliver antibody invivo.

Suitable regulatory sequences may be derived from a variety of sources,including bacterial, fungal, viral, mammalian, or insect genes.

Examples of such regulatory sequences include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the host cell chosen and the vector employed,other sequences, such as an origin of replication, additional DNArestriction sites, enhancers, and sequences conferring inducibility oftranscription may be incorporated into the expression vector.

In an embodiment, the regulatory sequences direct or increase expressionin neural tissue and/or cells.

In an embodiment, the vector is a viral vector.

The recombinant expression vectors may also contain a marker gene whichfacilitates the selection of host cells transformed, infected ortransfected with a vector for expressing an antibody or epitope peptidedescribed herein.

The recombinant expression vectors may also contain genes which encode afusion moiety (i.e. a “fusion protein”) which provides increasedexpression or stability of the recombinant peptide; increased solubilityof the recombinant peptide; and aid in the purification of the targetrecombinant peptide by acting as a ligand in affinity purification,including for example tags and labels described herein. Further, aproteolytic cleavage site may be added to the target recombinant proteinto allow separation of the recombinant protein from the fusion moietysubsequent to purification of the fusion protein. Typical fusionexpression vectors include pGEX (Amrad Corp., Melbourne, Australia),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the recombinant protein.

Systems for the transfer of genes for example into neurons and neuraltissues both in vitro and in vivo include vectors based on viruses, mostnotably Herpes Simplex Virus, Adenovirus, Adeno-associated virus (AAV)and retroviruses including lentiviruses. Alternative approaches for genedelivery include the use of naked, plasmid DNA as well as liposome—DNAcomplexes. Another approach is the use of AAV plasmids in which the DNAis polycation-condensed and lipid entrapped and introduced into thebrain by intracerebral gene delivery (Leone et al. US Application No.2002076394).

Accordingly in another aspect, the compounds, immunogens, nucleic acids,vectors and antibodies described herein may be formulated in vesiclessuch as liposomes, nanoparticles, and viral protein particles, forexample for delivery of antibodies, compounds, immunogens and nucleicacids described herein. In particular synthetic polymer vesicles,including polymersomes, can be used to administer antibodies.

Also provided in another aspect is a cell, optionally an isolated and/orrecombinant cell, expressing an antibody described herein or comprisingan expression cassette or vector herein disclosed.

The recombinant cell can be generated using any cell suitable forproducing a polypeptide, for example suitable for producing an antibodyand/or binding fragment thereof. For example to introduce a nucleic acid(e.g. a vector) into a cell, the cell may be transfected, transformed orinfected, depending upon the vector employed.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the peptides and antibodies described hereinmay be expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus), yeast cells or mammalian cells.

In an embodiment, the cell is a eukaryotic cell selected from a yeast,plant, worm, insect, avian, fish, reptile and mammalian cell.

In an embodiment, the cell is a fused cell such as a hybridoma cell.

In another embodiment, the mammalian cell is a myeloma cell, a spleencell, or a hybridoma cell.

In an embodiment, the cell is a neural cell.

Yeast and fungi host cells suitable for expressing an antibody orpeptide include, but are not limited to Saccharomyces cerevisiae,Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces andvarious species of the genus Aspergillus. Examples of vectors forexpression in yeast S. cerivisiae include pYepSec1, pMFa, pJRY88, andpYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for thetransformation of yeast and fungi are well known to those of ordinaryskill in the art.

Mammalian cells that may be suitable include, among others: COS (e.g.,ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No.CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1cells. Suitable expression vectors for directing expression in mammaliancells generally include a promoter (e.g., derived from viral materialsuch as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), aswell as other transcriptional and translational control sequences.Examples of mammalian expression vectors include pCDM8 and pMT2PC.

A further aspect is a hybridoma cell line producing an antibody specificfor an epitope described herein.

Compositions

A further aspect is a composition comprising a compound, immunogen,nucleic acid, vector or antibody described herein.

In an embodiment, the composition comprises a diluent.

Suitable diluents for nucleic acids include but are not limited towater, saline solutions and ethanol.

Suitable diluents for polypeptides, including antibodies or fragmentsthereof and/or cells include but are not limited to saline solutions, pHbuffered solutions and glycerol solutions or other solutions suitablefor freezing polypeptides and/or cells.

In an embodiment, the composition is a pharmaceutical compositioncomprising any of the peptides, immunogens, antibodies, nucleic acids orvectors disclosed herein, and optionally comprising a pharmaceuticallyacceptable carrier.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionsthat can be administered to subjects, optionally as a vaccine, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle.

Pharmaceutical compositions include, without limitation, lyophilizedpowders or aqueous or non-aqueous sterile injectable solutions orsuspensions, which may further contain antioxidants, buffers,bacteriostats and solutes that render the compositions substantiallycompatible with the tissues or the blood of an intended recipient. Othercomponents that may be present in such compositions include water,surfactants (such as Tween), alcohols, polyols, glycerin and vegetableoils, for example. Extemporaneous injection solutions and suspensionsmay be prepared from sterile powders, granules, tablets, or concentratedsolutions or suspensions. The composition may be supplied, for examplebut not by way of limitation, as a lyophilized powder which isreconstituted with sterile water or saline prior to administration tothe patient.

Pharmaceutical compositions may comprise a pharmaceutically acceptablecarrier. Suitable pharmaceutically acceptable carriers includeessentially chemically inert and nontoxic compositions that do notinterfere with the effectiveness of the biological activity of thepharmaceutical composition. Examples of suitable pharmaceutical carriersinclude, but are not limited to, water, saline solutions, glycerolsolutions, ethanol, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammoniumchloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), andliposomes. Such compositions should contain a therapeutically effectiveamount of the compound, together with a suitable amount of carrier so asto provide the form for direct administration to the patient.

The composition may be in the form of a pharmaceutically acceptable saltwhich includes, without limitation, those formed with free amino groupssuch as those derived from hydrochloric, phosphoric, acetic, oxalic,tartaric acids, etc., and those formed with free carboxyl groups such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol,histidine, procaine, etc.

In an embodiment comprising a compound or immunogen described herein,the composition comprises an adjuvant.

Adjuvants that can be used for example, include Intrinsic adjuvants(such as lipopolysaccharides) normally are the components of killed orattenuated bacteria used as vaccines. Extrinsic adjuvants areimmunomodulators that are typically non-covalently linked to antigensand are formulated to enhance the host immune responses. Aluminumhydroxide, aluminum sulfate and aluminum phosphate (collectivelycommonly referred to as alum) are routinely used as adjuvants. A widerange of extrinsic adjuvants can provoke potent immune responses toimmunogens. These include saponins such as Stimulons (QS21, Aquila,Worcester, Mass.) or particles generated therefrom such as ISCOMs and(immunostimulating complexes) and ISCOMATRIX, complexed to membraneprotein antigens (immune stimulating complexes), pluronic polymers withmineral oil, killed mycobacteria and mineral oil, Freund's completeadjuvant, bacterial products such as muramyl dipeptide (MDP) andlipopolysaccharide (LPS), as well as lipid A, and liposomes.

In an embodiment, the adjuvant is aluminum hydroxide. In anotherembodiment, the adjuvant is aluminum phosphate. Oil in water emulsionsinclude squalene; peanut oil; MF59 (WO 90/14387); SAF (SyntexLaboratories, Palo Alto, Calif.); and Ribi™ (Ribi Immunochem, Hamilton,Mont.). Oil in water emulsions may be used with immunostimulating agentssuch as muramyl peptides (for example,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide(TM)), or other bacterial cell wallcomponents.

The adjuvant may be administered with an immunogen as a singlecomposition. Alternatively, an adjuvant may be administered before,concurrent and/or after administration of the immunogen.

Commonly, adjuvants are used as a 0.05 to 1.0 percent solution inphosphate—buffered saline. Adjuvants enhance the immunogenicity of animmunogen but are not necessarily immunogenic themselves. Adjuvants mayact by retaining the immunogen locally near the site of administrationto produce a depot effect facilitating a slow, sustained release ofimmunogen to cells of the immune system. Adjuvants can also attractcells of the immune system to an immunogen depot and stimulate suchcells to elicit immune responses. As such, embodiments encompasscompositions further comprising adjuvants.

Adjuvants for parenteral immunization include aluminum compounds (suchas aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate). The antigen can be precipitated with, or adsorbed onto, thealuminum compound according to standard protocols. Other adjuvants suchas RIBI (ImmunoChem, Hamilton, Mont.) can also be used in parenteraladministration.

Adjuvants for mucosal immunization include bacterial toxins (e.g., thecholera toxin (CT), the E. coli heat-labile toxin (LT), the Clostridiumdifficile toxin A and the pertussis toxin (PT), or combinations,subunits, toxoids, or mutants thereof). For example, a purifiedpreparation of native cholera toxin subunit B (CTB) can be of use.Fragments, homologs, derivatives, and fusion to any of these toxins arealso suitable, provided that they retain adjuvant activity. Preferably,a mutant having reduced toxicity is used. Suitable mutants have beendescribed (e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627(Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PTmutant)). Additional LT mutants that can be used in the methods andcompositions include, for example Ser-63-Lys, Ala-69-Gly, Glu-110-Asp,and Glu-112-Asp mutants. Other adjuvants (such as a bacterialmonophosphoryl lipid A (MPLA) of various sources (e.g., E. coli,Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri,saponins, or polylactide glycolide (PLGA) microspheres) can also be usedin mucosal administration.

Other adjuvants include cytokines such as interleukins for example IL-1,IL-2 and IL-12, chemokines, for example CXCL10 and CCL5, macrophagestimulating factor, and/or tumor necrosis factor. Other adjuvants thatmay be used include CpG oligonucleotides (Davis. Curr Top MicrobiolImmunol., 247:171-183, 2000).

Oil in water emulsions include squalene; peanut oil; MF59 (WO 90/14387);SAF (Syntex Laboratories, Palo Alto, Calif.); and Ribi™ (RibiImmunochem, Hamilton, Mont.). Oil in water emulsions may be used withimmunostimulating agents such as muramyl peptides (for example,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) theramide(TM)), or other bacterial cell wallcomponents.

Adjuvants useful for both mucosal and parenteral immunization includepolyphosphazene (for example, WO 95/2415), DC-chol (3b-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol (for example,U.S. Pat. No. 5,283,185 and WO 96/14831) and QS-21 (for example, WO88/9336).

An adjuvant may be coupled to an immunogen for administration. Forexample, a lipid such as palmitic acid, may be coupled directly to oneor more peptides such that the change in conformation of the peptidescomprising the immunogen does not affect the nature of the immuneresponse to the immunogen.

The adjuvant may be administered with an immuogen as a singlecomposition. Further, an adjuvant may be administered before, concurrentor after administration of the immunogen.

In an embodiment, the composition comprises an antibody describedherein. In another embodiment, the composition comprises an antibodydescribed herein and a diluent. In an embodiment, the composition is asterile composition.

III. Kits

A further aspect relates to a kit comprising i) an antibody and/orbinding fragment thereof, ii) a nucleic acid, iii) composition or iv)recombinant cell described herein, comprised in a vial such as a sterilevial or other housing and optionally a reference agent and/orinstructions for use thereof.

In an embodiment, the kit further comprises one or more of a collectionvial, standard buffer and detection reagent.

IV. Methods

Included are methods for making the compounds, immunogens and antibodiesdescribed herein.

In particular, provided are methods of making an antibody selective fora conformational epitope of QKLV (SEQ ID NO: 1) or related epitopecomprising administering to a subject, optionally a non-human subject, aconformationally restricted compound comprising an epitope sequencedescribed herein, optionally cyclic compound comprising QKLV (SEQ IDNO: 1) or related epitope, and isolating antibody producing cells orantibodies that specifically or selectively bind the cyclic compound andoptionally i) specifically or selectively bind synthetic and/or nativeoligomers and/or that have no or negligible senile plaque binding insitu tissue samples or no or negligible binding to a correspondinglinear peptide. The cyclic compound can for example comprise any of the“epitopes” described herein containing cyclic compounds describedherein.

In an embodiment, the method is for making a monoclonal antibody usingfor example a method as described herein.

In an embodiment, the method is for making a humanized antibody usingfor example a method described herein.

Antibodies produced using a cyclic compound are selected as describedherein and in the Examples. In an embodiment, the method comprisesisolating antibodies that specifically or selectively bind cyclicpeptide over linear peptide, are specific for the epitope sequence,specifically bind oligomer and/or lack or negligibly bind plaque in situand/or corresponding linear peptide, optionally using a method describedherein.

A further aspect provides a method of detecting whether a biologicalsample comprises A-beta the method comprising contacting the biologicalsample with an antibody described herein and detecting the presence ofany antibody complex. In an embodiment, the method is for detectingwhether a biological sample comprises A-beta. In an embodiment themethod is for detecting whether the sample comprises A-beta wherein QKLV(SEQ ID NO: 1) or a related epitope is in an alternate conformation forexample wherein at least Q, L or V is in an alternate conformation thanoccupied by Q, L or V in a non-oligomeric conformation.

In an embodiment, the method comprises:

a. contacting the biologic sample with an antibody described herein thatis specific and/or selective for A-beta oligomer herein under conditionspermissive to produce an antibody:A-beta oligomer complex; and

b. detecting the presence of any complex;

wherein the presence of detectable complex is indicative that the samplemay contain A-beta oligomer.

In an embodiment, the level of complex formed is compared to a testantibody such as a suitable Ig control or irrelevant antibody.

In an embodiment, the detection is quantitated and the amount of complexproduced is measured. The measurement can for example be relative to astandard.

In an embodiment, the measured amount is compared to a control.

In another embodiment, the method comprises:

(a) contacting a biological sample of said subject with an antibodydescribed herein, under conditions permissive to produce anantibody-antigen complex;

(b) measuring the amount of the antibody-antigen complex in the testsample; and

(c) comparing the amount of antibody-antigen complex in the test sampleto a control;

wherein detecting antibody-antigen complex in the biological sample ascompared to the control indicates that the sample comprises A-beta.

The control can be a sample control (e.g. from a subject without AD, orfrom a subject with a particular form of AD, mild, moderate oradvanced), or be a previous sample from the same subject for monitoringchanges in A-beta oligomer levels in the subject.

In an embodiment, an antibody described herein is used.

In an embodiment, the A-beta comprising a QKLV (SEQ ID NO: 1)conformational epitope is A-beta oligomer, wherein the detecting thepresence of antigen:antibody complex is indicative that the sample maycontain A-beta oligomer.

In an embodiment, the antibody specifically and/or selectivelyrecognizes a conformation of A-beta comprising a QKLV (SEQ ID NO: 1) orrelated conformational epitope, and detecting the antibody antigencomplex in the biological sample is indicative that sample comprisesA-beta oligomer.

In an embodiment, the sample is a biological sample. In an embodiment,the sample comprises brain tissue or an extract thereof and/or CSF. Inan embodiment, the sample comprises whole blood, plasma or serum. In anembodiment, the sample is obtained from a human subject. In anembodiment, the subject is suspected of, at a risk of or has AD.

A number of methods can be used to detect an A-beta:antibody complex andthereby determine if A-beta comprising a QKLV (SEQ ID NO: 1) or relatedconformational epitope and/or A-beta oligomers is present in thebiological sample using the antibodies described herein, includingimmunoassays such as flow cytometry, Western blots, ELISA, andimmunoprecipitation followed by SDS-PAGE immunocytochemistry.

As described in the Examples surface plasmon resonance technology can beused to assess conformation specific binding. If the antibody is labeledor a detectably labeled secondary antibody specific for the complexantibody is used, the label can be detected. Commonly used reagentsinclude fluorescent emitting and HRP labeled antibodies. In quantitativemethods, the amount of signal produced can be measured by comparison toa standard or control. The measurement can also be relative.

A further aspect includes a method of measuring a level of or imagingA-beta in a subject or tissue, optionally where the A-beta to bemeasured or imaged is oligomeric A-beta. In an embodiment, the methodcomprises administering to a subject at risk or suspected of having orhaving AD, an antibody conjugated to a detectable label; and detectingthe label, optionally quantitatively detecting the label. The label inan embodiment is a positron emitting radionuclide which can for examplebe used in PET imaging.

A further aspect includes a method of inducing an immune response in asubject, comprising administering to the subject a compound describedherein, optionally a cyclic compound comprising QKLV (SEQ ID NO: 1) or arelated epitope peptide sequence, an immunogen and/or compositioncomprising said compound or said immunogen; and optionally isolatingcells and/or antibodies that specifically and/or selectively bind theA-beta peptide in the compound or immunogen administered. In anembodiment, the composition is a pharmaceutical composition comprisingthe compound or immunogen in admixture with a pharmaceuticallyacceptable, diluent or carrier.

In an embodiment, the subject is a non-human subject such as a rodent.Antibody producing cells generated are used in an embodiment to producea hybridoma cell line.

In an embodiment, the immunogen administered comprises a compound ofFIG. 12.

It is demonstrated herein that antibodies raised against cycloCGQKLVG(SEQ ID NO: 2), can specifically and/or selectively bind A-betaoligomers and lack A-beta plaque staining. Oligomeric A-beta species arebelieved to be the toxic propagating species in AD. Further as shown inFIG. 21, antibodies raised using cyclo(CGQKLVG) (SEQ ID NO: 3) andspecific for oligomers, inhibited A-beta aggregation and A-beta oligomerpropagation. In addition, such antibodies inhibited the toxicity ofA-beta oligomers on neural cells in an in vitro assay (FIG. 22)Accordingly, also provided are methods of inhibiting A-beta oligomerpropagation, the method comprising contacting a cell or tissueexpressing A-beta with or administering to a subject in need thereof aneffective amount of an A-beta oligomer specific or selective antibodydescribed herein to inhibit A-beta aggregation and/or oligomerpropagation. In vitro the assay can be monitored as described in Example10.

The antibodies may also be useful for treating AD and/or other A-betaamyloid related diseases. For example, variants of Lewy body dementiaand in inclusion body myositis (a muscle disease) exhibit similarplaques as AD and A-beta can also form aggregates implicated in cerebralamyloid angiopathy. As mentioned, antibodies raised to cyclo(CGQKLVG)(SEQ ID NO: 3) bind oligomeric A-beta which is believed to be atoxigenic species of A-beta in AD and inhibit formation and propagationof toxigenic A-beta oligomers.

Accordingly a further aspect is a method of treating AD and/or otherA-beta amyloid related diseases, the method comprising administering toa subject in need thereof i) an effective amount of an antibodydescribed herein, optionally an A-beta oligomer specific or selective ora pharmaceutical composition comprising said antibody; or 2)administering an isolated cyclic compound comprising QKLV (SEQ ID NO: 1)or a related epitope sequence or immunogen or pharmaceutical compositioncomprising said cyclic compound, to a subject in need thereof.

In an embodiment, a biological sample from the subject to be treated isassessed for the presence or levels of A-beta using an antibodydescribed herein. In an embodiment, a subject with detectable A-betalevels (e.g. A-beta antibody complexes measured in vitro or measured byimaging) is treated with the antibody.

The antibody and immunogens can for example be comprised in apharmaceutical composition as described herein, and formulated forexample in vesicles for improving delivery.

One or more antibodies targeting cycloCGQKLVG (SEQ ID NO: 3) and/orrelated antibodies can be administered in combination. In addition theantibodies disclosed herein can be administered with one or more othertreatments such as a beta-secretase inhibitor or a cholinesteraseinhibitor.

In an embodiment, the antibody is a conformation specific/selectiveantibody, optionally that specifically or selectively binds A-betaoligomer.

Also provided are uses of the compositions, antibodies, isolatedpeptides, immunogens and nucleic acids for treating AD.

The compositions, compounds, antibodies, isolated peptides, immunogensand nucleic acids, vectors etc. described herein can be administered forexample, by parenteral, intravenous, subcutaneous, intramuscular,intracranial, intraventricular, intrathecal, intraorbital, ophthalmic,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol ororal administration.

In certain embodiments, the pharmaceutical composition is administeredsystemically.

In other embodiments, the pharmaceutical composition is administereddirectly to the brain or other portion of the CNS. For example suchmethods include the use of an implantable catheter and a pump, whichwould serve to discharge a pre-determined dose through the catheter tothe infusion site. A person skilled in the art would further recognizethat the catheter may be implanted by surgical techniques that permitvisualization of the catheter so as to position the catheter adjacent tothe desired site of administration or infusion in the brain. Suchtechniques are described in Elsberry et al. U.S. Pat. No. 5,814,014“Techniques of Treating Neurodegenerative Disorders by Brain Infusion”,which is herein incorporated by reference. Also contemplated are methodssuch as those described in US patent application 20060129126 (Kaplittand During “Infusion device and method for infusing material into thebrain of a patient”. Devices for delivering drugs to the brain and otherparts of the CNS are commercially available (eg. SynchroMed® EL InfusionSystem; Medtronic, Minneapolis, Minn.)

In another embodiment, the pharmaceutical composition is administered tothe brain using methods such as modifying the compounds to beadministered to allow receptor-mediated transport across the blood brainbarrier.

Other embodiments contemplate the co-administration of the compositions,compounds, antibodies, isolated peptides, immunogens and nucleic acidsdescribed herein with biologically active molecules known to facilitatethe transport across the blood brain barrier.

Also contemplated in certain embodiments, are methods for administeringthe compositions, compounds, antibodies, isolated peptides, immunogensand nucleic acids described herein across the blood brain barrier suchas those directed at transiently increasing the permeability of theblood brain barrier as described in U.S. Pat. No. 7,012,061 “Method forincreasing the permeability of the blood brain barrier”, hereinincorporated by reference.

A person skilled in the art will recognize the variety of suitablemethods for administering the compositions, compounds, antibodies,isolated peptides, immunogens and nucleic acids described hereindirectly to the brain or across the blood brain barrier and be able tomodify these methods in order to safely administer the productsdescribed herein.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

EXAMPLES Example 1

I. Gō Model Method for Predicting A-Beta Oligomer Specific Epitopes

One epitope prediction model is based on the free energy landscape ofpartial protein unfolding from the native state. The native state istaken to be an experimentally-derived fibril structure. When the proteinis partially unfolded from the native state by a given amount of primarysequence, epitope candidates are contiguous sequence segments that costthe least free energy to disorder. The free energy of a given proteinconformation arises from several contributions, including conformationalentropy and solvation of polar functional groups that favor the unfoldedstate, as well as the loss of electrostatic and van der Waalsintra-protein interactions that enthalpically stabilize the nativestate.

A. Gō-Like Model of Protein Partially Unfolding Landscape

An approximate model to account for the free energetic changes that takeplace during unfolding assigns a fixed energy to all contacts in thenative state, where a contact is defined as a pair of heavy(non-hydrogen) atoms within a fixed cut-off distance r_(cutoff). Go-likemodels have been successfully implemented in previous studies of proteinfolding. The Gō-like model isolates the effects arising from thetopology of native protein interactions, and in practice the unfoldingfree energy landscape can be readily calculated from a single nativestate structure.

The total free energy cost of unfolding a segment depends on the numberof interactions to be disrupted, together with the conformationalentropy term of the unfolded region.

In the following equations, lower case variables refer to atoms, whileupper case variables refer to residues. Let T be the set of all residuesin the protein, U be the set of residues unfolded in the protein, and Fbe the subset of residues folded in the protein (thus T=U ∪F). Theunfolding mechanism at high degrees of nativeness consists of multiplecontiguous strands of disordered residues. Here the approximation of asingle contiguous unfolded strand was adopted, and the free energy costto disorder this contiguous strand was calculated.

The total free energy change ΔF_(Gō)(U) for unfolding the set ofresidues U isΔF _(Gō)(U)=E_(Gō)(U)−TΔS _(Gō)(U)  (1)

The unfolding enthalpy function ΔE_(Gō)(U) is given by the number ofinteractions disrupted by unfolding of the set of U residues:

$\begin{matrix}{{\Delta\;{E_{G\;\overset{\_}{o}}{()}}} = {a\;\Theta\;( {r_{cutoff} - {{r_{i} - r_{j}}}} )}} & (2)\end{matrix}$

In Equation 2, the sum on i, j is over all unique pairs of heavy atomsthat have either one or both atoms in the unfolded region, r_(i) andr_(j) are the coordinates of atoms i and j, r_(cutoff) (taken to be 4.8Å) is the interaction distance cut-off. ⊖(x) is the Heaviside functiondefined by ⊖(x)=1 if x is positive and 0 otherwise. The energy percontact a may be chosen to recapitulate the overall experimentalstability ΔF_(Exp)(U)|_(u=T) on completely unfolding the protein at roomtemperature:

$\begin{matrix}{a = \frac{ {\Delta\;{F_{E\;{xp}}{()}}} \middle| {}_{=}{{+ T}\;\Delta\;{S_{G\;\overset{\_}{o}}{()}}} |_{=}}{\sum_{i,{j \in}}^{i > j}{\Theta( {r_{cutoff} - {{r_{i} - r_{j}}}} )}}} & (3)\end{matrix}$

The results do not depend on this value; it merely sets the overallglobal energy scale in the problem. In the present model, this freeenergy was taken to be a constant number equal to 4.6 kcal/mol. Thisvalue is not a primary concern as it is the relative free energy costfor the different regions of the same protein that is sought to bedisordered in the method of epitope prediction.

The calculation of the unfolding entropy term ΔS_(Gō)(U) is discussed inB below.

B. Entropy Calculation

The number of microstates accessible to the protein in the unfoldedstate is much greater than the number accessible in the native state, sothere is a favorable gain of conformational entropy on unfolding. Thetotal entropy of the unfolding segment U by summing over all theresidues K in the unfolded region is calculated

Δ ⁢ ⁢ S G ⁢ ⁢ o _ ⁡ ( ) = ⁢ ( Δ ⁢ ⁢ S bb , K - ( 1 - ) ⁢ Δ ⁢ ⁢ S bu → ex , K + Δ ⁢ ⁢S ex → sol , K ) ( 4 )where ΔS_(bb,K), ΔS_(bu→ex,K), ΔS_(ex→sol,K) are the threeconformational entropic components of residue K as listed in reference[3]: ΔS_(bb,K) is the backbone entropy change from native state tounfolded state, ΔS_(bu→ex,K) is the entropy change for side-chain fromburied inside protein to the surface of the protein, and, andΔS_(ex→sol,K) is the entropy obtained for the side-chain from thesurface to the solution.

A correction is applied to the unfolded state conformational entropies,since in the single sequence approximation the end points of thepartially unfolded strand are fixed in their positions in the nativestructure. This means that there is a loop entropy penalty to be paidfor constraining the ends in the partially unfolded structure, which isnot present in the fully unfolded stateS _(return) =−k _(B) ln(f _(w)(R|N)ΔT  (5)

Here f_(w)(R|N)_(ΔT) is found by calculating the probability an idealrandom walk returns to a box of volume ΔT centered at position R after Nsteps, without penetrating back into the protein during the walk. Forstrand lengths shorter than about n≈20 residues, the size of the meltedstrand is much smaller than the protein diameter and the steric excludedvolume of the protein is well treated as an impenetrable plane. Thenumber of polymeric states of the melted strand must be multiplied bythe fraction of random walks that travel from an origin on the surfaceof the protein to a location where the melted polymer re-enters theprotein without touching or crossing the impenetrable plane. The abovefraction of states can be written in the following form:

$\begin{matrix}{{f_{w}( R \middle| N )} = {\frac{a}{N^{5/2}}{\exp( {{- \frac{3R^{2}}{2{Nl}^{2}}} - \frac{N^{2}V_{c}}{2R^{3}}} )}}} & (6)\end{matrix}$where R is the end to end distance between the exit and entrancelocations, N is the number of residues of the melted region, and a, I,V_(C) are parameters determined by fitting to unfolded polypeptidesimulations. The parameter I is the effective arc length between twoC_(α) atoms, and V_(C) is the average excluded volumes for each residue.By fitting the Equation 6 into the simulation results, the values of theparameters a=0.0217, I=4.867, V_(C)=3.291 are obtained. This entropypenalty is general and independent of the sequence.

Disulfide bonds require additional consideration in the loop entropyterm since they further restrict the motion of the unfolded segment.When present, the disulfide is treated as an additional node throughwhich the loop must pass, in effect dividing the full loop into twosmaller loops both subject to the boundary conditions described above.

C. Epitope Prediction from Free Energy Landscape

Once the free energy landscape of partially unfolding the protein isobtained, a variable energy threshold E_(th) is applied, and thesegments that contains no fewer than 3 amino acids and with free energycost below the threshold are predicted as epitope candidates. Theprediction is stable with respect to varying the threshold value E_(th).

II. Epitopes Predictions from A-Beta Disrupted Fibril Structures

From the native structure in 2M4J, the unfolding free energy landscapeusing the Gō-like model as described in I A was calculated. The resultis shown in FIG. 1. The epitope QKLV (SEQ ID NO: 1) emerges as acandidate epitope in this analysis.

The free energy cost in cal/mol is shown, for all epitopes with freeenergy cost less than 10 kcal/mol. The epitope marked with a box givingthe x,y,z values is centered at residue ID 17 (x-axis), and has length 4(y-axis), thus consisting of residue 15-18 or QKLV (SEQ ID NO: 1). Foreven-numbered epitopes, the “center” is defined by convention as theresidue to the right of the numerical center, simply for purposes ofplotting the figure.

FIG. 1 shows the top view of the free energy landscape below a thresholdEth=10 kcal/mol, where the x-axis is the center of the segment, y-axisis the length of the segment, and the depth of color represents the freeenergy needed to melt the corresponding segment. From FIG. 1, at theenergy scale of 10 kcal/mol, the epitope consisting of residues 15-18 orsequence QKLV (SEQ ID NO: 1) emerges as a predicted epitope in chains A,B and C of the structure PDB 2M4J.

The fibril model contains chains “A” to “I”. For example, the determinedstructure of the fibril consists of a repeating unit of 9 chains, whichare identified as chains A-I. The identified epitope presents for chainsA, B, and C.

Likewise for structure A-beta fibril structure 2MXU, an epitope ofsequences 14-18 emerges for chain A (sequence HQKLV; FIG. 2). HQKLV (SEQID NO: 2) is consistent with an increased solvent exposure of Q and/orQK in the alternate conformation compared to monomeric or fibrillaryA-beta.

Example 2

Iii. Collective Coordinates Predictions

A second method for predicting misfolded epitopes is provided by amethod referred to as “Collective Coordinates biasing” which isdescribed in U.S. Patent Application Ser. No. 62/253,044, SYSTEMS ANDMETHODS FOR PREDICTING MISFOLDED PROTEIN EPITOPES BY COLLECTIVECOORDINATE BIASING filed Nov. 9, 2015, and is incorporated herein byreference. As described therein, the method usesmolecular-dynamics-based simulations which impose a global coordinatebias on a protein (or peptide-aggregate) to force the protein (orpeptide-aggregate) to misfold and then predict the most likely unfoldedregions of the partially unstructured protein (or peptide aggregate).Biasing simulations were performed and the solvent accessible surfacearea (SASA) corresponding to each residue index (compared to that of theinitial structure of the protein under consideration). SASA represents asurface area that is accessible to H2O. A positive change in SASA(compared to that of the initial structure of the protein underconsideration) may be considered to be indicative of unfolding in theregion of the associated residue index. The method was applied to threeA-beta strains, each with its own morphology: a three-fold symmetricstructure of Aβ-40 peptides (or monomers) (PDB entry 2M4J), a two-foldsymmetric structure of Aβ-40 monomers (PDB entry 2LMN), and asingle-chain, parallel in-register (e.g. a repeated beta sheet where theresidues from one chain interact with the same residues from theneighboring chains) structure of Aβ-42 monomers (PDB entry 2MXU).

Simulations were performed for each initial structure using thecollective coordinates method as described in U.S. Patent ApplicationSer. No. 62/253,044, SYSTEMS AND METHODS FOR PREDICTING MISFOLDEDPROTEIN EPITOPES BY COLLECTIVE COORDINATE BIASING and the CHARMMforce-field parameters described in: K. Vanommeslaeghe, E. Hatcher, C.Acharya, S. Kundu, S. Zhong, J. Shim, E. Darian, O. Guvench, P. Lopes,I. Vorobyov, and A. D. Mackerell. Charmm general force field: A forcefield for drug-like molecules compatible with the charmm all-atomadditive biological force fields. Journal of Computational Chemistry,31(4):671-690, 2010; and P. Bjelkmar, P. Larsson, M. A. Cuendet, B.Hess, and E. Lindahl. Implementation of the CHARMM force field inGROMACS: analysis of protein stability effects from correlation maps,virtual interaction sites, and water models. J. Chem. Theo. Comp.,6:459-466, 2010, both of which are hereby incorporated herein byreference, with TIP3P water.

The SASA of amino acid side chains as a function of residue index, forone monomer of the 3 fold symmetric A-beta structure 2M4J, after biasingto 80% of its initial structure was analysed. One region that emergedwith reliably increased SASA was residues 14-17, corresponding toresidues HQKL, and shown in FIG. 8. HQKL is consistent with an increasedsolvent exposure of Q and/or QK in the alternate conformation comparedto monomeric or fibrillary A-beta.

An epitope consisting of residues 14-17 was predicted for the 3-foldA-beta40 (2M4J) and 2 fold A-beta 40 (2LMN) structures.

An epitope consisting of residues 15-19 was predicted from A-beta40(2MXU) and an epitope consisting of residues 15-20 was predicted forA-beta42 constrained ends.

Different biasing demonstrates that the general structure of thepredicted epitopes does depend significantly on the degree of biasing.

FIG. 13 is a plot of the entropy with respect to the fibril for both alinear peptide CGQKLVG (SEQ ID NO: 3) and a cyclic peptidecyclo(CGQKLVG) (SEQ ID NO: 3). As shown therein the side chain entropyis reduced for the cyclic compound as compared to the linear peptide,indicating that the side-chains are more conformationally-constrainedthan when the side-chains are in the linear peptide, i.e. than in freeA-beta monomer.

Example 3

Constrained peptide structures comprising QKLV (SEQ ID NO: 1) may mimicthe conformational epitope identified using the prediction programsdescribed. Cyclic structures were designed and assessed includingstructures shown in FIG. 12. The cyclic structure shown in FIG. 12A wasassessed as described in Example 4.

Example 4

Determination of Structures Comprising the Conformational Epitope QKLV(SEQ ID NO: 1) that Approximates its Predicted Orientation in A-BetaOligomers

A cyclic compound comprising the QKLV (SEQ ID NO: 1) epitope (as shownin FIG. 5A) was assessed for providing an epitope with conformationalrelatedness or dissimilarity to the epitope orientation in A-beta fibriland monomers.

I. Curvature of the Cyclic Peptide

The curvature of the cyclic peptide compound backbone shown in FIG.12(a) has a profile different—it is larger—from either that of thefibril or that of the monomer, implying that an antibody directedagainst the cyclic peptide may show selectivity for a species presentinga different conformational ensemble than that of either the monomer orfibril. The curvature profiles are shown in FIG. 3.

FIG. 3 is a plot showing curvature as a function of residue index. Theaverage curvature in the equilibrium ensembles for the cyclic peptideCGQKLVG (SEQ ID NO: 3) is shown (long arrow), along with the curvaturefor the linear peptide (short arrow), and the curvature of the variousmonomers in the fibril (thin curves length of peptide), and the averagecurvature for the fibril (thick curve length of peptide).

The curvature as used herein refers to the backbone curvature and isdefined as the rate of change of the tangent vector as one moves alongthe backbone. This can be quantified by taking the unit tangent vectorbetween consecutive C_alpha atoms and then noting how it changes fromone tangent to the next. The precise mathematical definition used hereis a discrete version of the traditional definition of the curvature ofa space curve in differential geometry: K_(i+1)-arccos(t_(i)·t_(i+1)),where t, is the unit tangent vector from C_alpha(i) to C_alpha(i+1). Thecurvature is then simply an angle in radians that may vary in principlefrom 0 (parallel) to π (antiparallel), depending on the backboneconfiguration.

The values of the curvature were determined for Q, K, L, V incyclo(CGQKLVG) (SEQ ID NO: 3), linear CGQKLVG (SEQ ID NO: 3), and QKLV(SEQ ID NO: 1) in the context of the fibril respectively as:

-   Cyclic peptide: 1.248 1.566 1.422 1.46-   Linear Peptide: 0.870 1.355 0.931 1.303-   Fibril: 0.740 1.159 0.796 1.188

The averages of these are:

-   Cyclic peptide: 1.42-   Linear peptide: 1.11-   Fibril: 0.97

For the plots of curvature, and dihedral angle distributions below, thedata are obtained from equilibrium simulations in explicit solvent (SPC)using the Charmm27. cyclic peptide ensemble: simulation time 1 ns,containing 500 frames linear peptide ensemble: simulation time 10 ns,containing 1000 frames 2m4j ensemble: 680 ps, containing 68 frames.

Because the curvature of the cyclic epitope is larger, a hypotheticalturn on the oligomer containing these residues would have a backboneorientation that is distinct from that in the fibril or monomer, howeverthe degree of curvature would not be unphysical—values of curvaturecharacterizing the cyclic peptide are obtained in several locations ofthe fibril.

II. Dihedral Angle Distributions

As further computational support that the identified epitope may definean oligomer-specifc epitope in A-beta, the dihedral angle distributionsin the cyclic peptide of FIG. 12 a) which may be a proxy for an exposedepitope in the oligomer—are substantially different from thecorresponding distributions in either the fibril or monomer.

Distributions of dihedral angles for Q15 are substantially different inthe cyclic peptide from either the monomer or fibril distributions (FIG.4A), for many of the side-chain dihedrals.

FIG. 4B shows an image for the cyclic peptide CGQKLVG (SEQ ID NO: 3);image taken from an equilibrium molecular dynamics simulation. In thisimage, the glutamine (Q) residue has sidechain rendered with the C_(α),C_(β), C_(x), and C_(δ) atoms shown as black beads, and thecorresponding dihedral bond shown in black. This dihedral hassignificantly different dihedral distribution in the cyclic peptide thanthe corresponding dihedral distributions explored by either the linearpeptide or by the residue in the context of the fibril (see FIG. 4A,panel Res-Q:CA-CB-CG-CD).

Likewise, dihedral distributions can be examined for K, L, and V. Thedihedral distributions are shown for K16 in FIG. 5. The differences forK are less apparent than they are for Q.

The dihedral distributions are shown for L17 in FIG. 6.

The dihedral distributions are shown for V18 in FIG. 7.

Though L17 shows some discrepancies from linear peptide distributions,particularly for dihedrals involving backbone atoms that differentiatelinear from cyclic conformations, the residue showing the mostsignificant difference from either linear or fibril distributions isQ15.

Q15-K16 for example in the context of QKL or QKLV may be key residues onthe epitope. Further support for the importance of these residues isprovided by a surface area and solubility analysis described herein.

III. Solubility and Antigenicity of the Epitope

The solubility of the residues of A-beta 42 according to the CamSolprediction scheme [4] is shown in the FIG. 9. Residues QKLV (SEQ IDNO: 1) in FIG. 9 have values of −0.899, −0.936, −1.46, and −1.51respectively. The solubility decreases as one continues towards theC-terminus.

The more soluble a residue is, the more likely it is to be encounteredon the surface of any species of A-beta, and in particular it is morelikely to be on or near the surface of the oligomer. A residue will besolvated on the surface of unstructured monomer, and may not be solvatedon the surface of fibrils (because of an organized structure). Oligomerswill be partially and dynamically structured so that the exposure ofspecific residues which are not solvent-accessible in the fibril,optionally in combination with one or more conformational features, maydistinguish the oligomer from the fibril.

A relative solubility factor σ_(i) for residue i, can be defined as

$\sigma_{i} = \frac{s_{i} - s_{m\; i\; n}}{s_{m\; a\; x} - s_{m\; i\; n}}$where s, is the solubility of residue i, and s_(max) and s_(min) are themaximum and minimum values of the solubility in a given range ofresidues. Here, simply to set a scale for plotting values, the range ofresidues is arbitrarily chosen to be HQKLVF (SEQ ID NO: 9), so thats_(max)=0.0302 and s_(min)=−1.51.

When SASA is weighted by the solubility of the residue, more emphasis isput on the N-terminal residues in the group QKLV (SEQ ID NO: 1). FIG. 10plots the solvent accessible surface area (SASA), the SASA weighted bythe solubility factor for each residue, σi·SASAi, and σi·SASAi minus thevalue in the fibril, i.e. the increase in this quantity in the monomerand cyclic peptide over the fibril, σi·ΔSASAi.

Weighting by the solubility results in the Q and K residues having themost likelihood of differential exposure and antigenicity on the surfaceof oligomers.

Dissection of Solvent-Accessible Surface Area Based on ResidueSide-Chain Moieties

Separating sidechains of the various residues into atomic groups, shownin FIG. 11, shows that there is a general trend towards larger solventexposure as one approaches the termini of the side-chains. E.g. Q15 issubdivided into C_(β)—H₂, C_(γ)—H₂, C_(δ)—O_(ϵ), and N_(ϵ)—H₂, with theN_(ε)—H₂ having the most SASA in the cyclic peptide as compared to thefibril or monomer. The deviation in solvent exposure w.r.t. the fibrilalso has a tendency to increase for moieties near the termini of theside chains.

Example 5

Cyclic Compound Construction Comprising a Conformationally ConstrainedEpitope

Peptides comprising QKLV (SEQ ID NO: 1) such as Cyclo(CGQKLVG) (SEQ IDNO: 3) can be cyclized head to tail.

A linear peptide comprising QKLV (SEQ ID NO: 1) and a linker, preferablycomprising 2, 3, or 4 amino acids and/or PEG units, can be synthesizedusing known methods such as Fmoc based solid phase peptide synthesisalone or in combination with other methods. PEG molecules can be coupledto amine groups at the N terminus for example using coupling chemistriesdescribed in Hamley 2014 [6] and Roberts et al 2012 [7], eachincorporated herein by reference. The linear peptide compound may becyclized by covalently bonding 1) the amino terminus and the carboxyterminus of the peptide+linker to form a peptide bond (e.g. cyclizingthe backbone), 2) the amino or carboxy terminus with a side chain in thepeptide+linker or 3) two side chains in the peptide+linker.

The bonds in the cyclic compound may be all regular peptide bonds(homodetic cyclic peptide) or include other types of bonds such asester, ether, amide or disulfide linkages (heterodetic cyclic peptide).

Peptides may be cyclized by oxidation of thiol- or mercaptan-containingresidues at the N-terminus or C-terminus, or internal to the peptide,including for example cysteine and homocysteine. For example twocysteine residues flanking the peptide may be oxidized to form adisulphide bond. Oxidative reagents that may be employed include, forexample, oxygen (air), dimethyl sulphoxide, oxidized glutathione,cystine, copper (II) chloride, potassium ferricyanide, thallium(III)trifluro acetate, or other oxidative reagents such as may be known tothose of skill in the art and used with such methods as are known tothose of skill in the art.

Methods and compositions related to cyclic peptide synthesis aredescribed in US Patent Publication 2009/0215172. US Patent publication2010/0240865, US Patent Publication 2010/0137559, and U.S. Pat. No.7,569,541 describe various methods for cyclization. Other examples aredescribed in PCT Publication WO01/92466, and Andreu et al., 1994.Methods in Molecular Biology 35:91-169.

More specifically, a cyclic peptide comprising the QKLV (SEQ ID NO: 1)epitope can be constructed by adding a linker comprising a spacer withcysteine residues flanking and/or inserted in the spacer. The peptidecan be structured into a cyclic conformation by creating a disulfidelinkage between the non-native cysteines residues added to the N- andC-termini of the peptide. It can also be synthesized into a cycliccompound by forming a peptide bond between the N- and C-termini aminoacids (e.g. head to tail cyclization).

Peptide synthesis is performed by CPC Scientific Inc. (Sunnyvale Calif.,USA) following standard manufacturing procedures.

For example Cyclo(CGQKLVC) cyclic peptide comprising the conformationalepitope QKLV (SEQ ID NO: 1) is constructed in a constrained cyclicconformation using a disulfide linkage between cysteine residues addedto the N- and C-termini of a peptide comprising QKLV (SEQ ID NO: 1). Twonon-native cysteine residues were added to GQKLV (SEQ ID NO: 4) one atthe C-terminus and one at the N-terminus. The two cysteines are oxidizedunder controlled conditions to form a disulfide bridge or reacted headto tail to produce a peptide bond.

As described above, the structure of the cyclic peptide was designed tomimic the conformation and orientation of the amino acid side changes ofQKLV (SEQ ID NO: 1) in A-beta oligomer.

Cyclo(CGQKLVG)

A linear peptide comprising spacer GCG and epitope peptide QKLV (SEQ IDNO: 1) is synthesized for example using Fmoc based solid-phase peptidesynthesis. The solid phase can be on a rink amide resin or on a2-chlorotrityl resin.

Cyclo(CGQKLVG) (SEQ ID NO: 3) can be prepared by amide condensation ofthe linear peptide CGQKLVG (SEQ ID NO: 3).

Cyclo(C-PEG2-QKLVG) can be prepared by amide condensation of the linearcompound C-PEG2-QKLVG.

Immunogen Construction

The cyclic compound containing the constrained epitope peptide isoptionally conjugated to an immunogenicity enhancing agent such asKeyhole Limpet Hemocyanin (KLH) or a carrier such bovine serum albumin(BSA) using for example the method described in Lateef et al 2007,herein incorporated by reference.

Example 6

Antibody Generation and Selection

A conformational constrained compound optionally a cyclic compound suchas a cyclic peptide comprising QKLV (SEQ ID NO: 1) such ascyclo(CGQKLVG) (SEQ ID NO: 3) peptide is linked to Keyhole LimpetHemocyanin (KLH). The cyclopeptide is sent for mouse monoclonal antibodyproduction (ImmunoPrecise Antibodies LTD (Victoria BC, Canada),following protocols approved by the Canadian Council on Animal Care.Mouse sera are screened using either the conformational peptide used forproducing the antibodies or a related peptide e.g. cyclo(CGQKLV-peptide)(SEQ ID NO: 3), linked to BSA. Positive IgG-secreting clones aresubjected to large-scale production.

Hybridomas were made using an immunogen comprising cyclo(CGQKLVG) (SEQID NO: 3) as further described in Example 8. Hybridoma supernatants werescreened by ELISA and SPR for preferential binding to cyclo(CGQKLVG)(SEQ ID NO: 3) peptide vs linear (unstructured) peptide as describedherein. Positive IgG-secreting clones are subjected to large-scaleproduction and further purification using Protein G.

Example 7

Assessing Binding or Lack thereof to Plaques/Fibrils

For immunostaining, antibodies described herein, positive control 6E10(1 μg/ml) and isotype control IgG1, IgG2a, IgG2b, or IgG3 (1 μg/ml,Abcam) are used as primary antibodies. Sections are incubated overnightat 4° C., and washed 3×5 min in TBS-T. Anti-Mouse IgG HorseradishPeroxidase conjugated (1:1000, ECL) is applied to sections and incubated45 min, then washed 3×5 min in TBS-T. DAB chromogen reagent (VectorLaboratories, Burlington ON, Canada) is applied and sections rinsed withdistilled water when the desired level of target to background stainingis achieved. Sections are counterstained with Mayer's haematoxylin,dehydrated and cover slips were applied. Slides are examined under alight microscope (Zeiss Axiovert 200M, Carl Zeiss Canada, Toronto ON,Canada) and representative images captured at 50, 200 and 400×magnification using a Leica DC300 digital camera and software (LeicaMicrosystems Canada Inc., Richmond Hill, ON).

Example 8

Methods and Materials

Immunogen

Cyclic and linear peptides were generated at CPC Scientific, Sunnyvale,Calif., USA. Peptides were conjugated to KLH (for immunizing) and BSA(for screening) using a trifluoroacetate counter ion protocol. Peptideswere desalted and checked by MS and HPLC and deemed 95% pure. Peptideswere shipped to IPA for use in production of monoclonal antibodies inmouse.

Antibodies

A number of hybridomas and monoclonal antibodies were generated tocyclo(CGQKLVG) (SEQ ID NO: 3) linked to Keyhole Limpet Hemocyanin (KLH).

Fifty day old female BALB/c mice (Charles River Laboratories, Quebec)were immunized. A series of subcutaneous aqueous injections containingantigen but no adjuvant were given over a period of 19 days. Mice wereimmunized with 100 μg of peptide per mouse per injection of a 0.5 mg/mLsolution in sterile saline of cyclic peptide-KLH. Mice were housed in aventilated rack system from Lab Products. All 4 mice were euthanized onDay 19 and lymphocytes were harvested for hybridoma cell linegeneration.

Fusion/Hybridoma Development

Lymphocytes were isolated and fused with murine SP2/0 myeloma cells inthe presence of poly-ethylene glycol (PEG 1500). Fused cells werecultured using HAT selection. This method uses a semi-solidmethylcellulose-based HAT selective medium to combine the hybridomaselection and cloning into one step. Single cell-derived hybridomas growto form monoclonal colonies on the semi-solid media. 10 days after thefusion event, resulting hybridoma clones were transferred to 96-welltissue culture plates and grown in HT containing medium until mid-loggrowth was reached (5 days).

Hybridoma Analysis (Screening)

Tissue culture supernatants from the hybridomas were tested by indirectELISA on screening antigen (cyclic peptide-BSA) (Primary Screening) andprobed for both IgG and IgM antibodies using a Goatanti-IgG/IgM(H&L)-HRP secondary and developed with TMB substrate.Clones >0.2 OD in this assay were taken to the next round of testing.Positive cultures were retested on screening antigen to confirmsecretion and on an irrelevant antigen (Human Transferrin) to eliminatenon-specific mAbs and rule out false positives. All clones of interestwere isotyped by antibody trapping ELISA to determine if they are IgG orIgM isotype. All clones of interest were also tested by indirect ELISAon other cyclic peptide-BSA conjugates as well as linear peptide-BSAconjugates to evaluate cross-reactivity.

Mouse hybridoma antibodies were screened by Indirect ELISA usingcyclo(CGQKLVG) (SEQ ID NO: 3) conjugated to BSA.

ELISA Antibody Screening

Briefly, the ELISA plates were coated with 0.1 μg/wellcyclo(CGQKLVG)—conjugated-BSA (SEQ ID NO: 3) at 100 uL/well in carbonatecoating buffer (pH 9.6) O/N at 4 C and blocked with 3% skim milk powderin PBS for 1 hour at room temperature. Primary Antibody: Hybridomasupernatant at 100 uL/well incubated for 1 hour at 37 C with shaking.Secondary Antibody 1:10,000 Goat anti-mouse IgG/IgM(H+L)-HRP at 100uL/well in PBS-Tween for 1 hour at 37 C with shaking. All washing stepswere performed for 30 mins with PBS-Tween. The substrate3,3′,5,5′-tetramethylbenzidine (TMB) was added at 50 uL/well, developedin the dark and stopped with equal volume 1M HCl.

Positive clones were selected for further testing. Positive clones ofmouse hybridomas were tested for reactivity to cyclo(CGQKLVG) (SEQ IDNO: 3) conjugated BSA and human transferrin (HT) by indirect ELISA.Plates were coated with 1) 0.1 ug/well cyclo(CGQKLVG)—conjugated-BSA(SEQ ID NO: 3) at 100 uL/well in carbonate coating buffer (pH 9.6) 0/Nat 4 C; or 2) 0.25 ug/well HT Antigen at 50 uL/well in dH2O/N at 37 C.Primary Antibody: Hybridoma supernatant at 100 uL/well incubated for 1hour at 37 C with shaking. Secondary Antibody 1:10,000 Goat anti-mouseIgG/IgM(H+L)-HRP at 100 uL/well in PBS-Tween for 1 hour at 37 C withshaking. All washing steps were performed for 30 mins with PBS-Tween.The substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was added at 50uL/well, developed in the dark and stopped with equal volume 1M HCl.

ELISA Cyclo vs Linear CGQKLVG (SEQ ID NO: 3) Compound Selectivity

ELISA plates were coated with 1) 0.1 ug/wellcyclo(CGQKLVG)—conjugated-BSA (SEQ ID NO:3) at 100 uL/well in carbonatecoating buffer (pH 9.6) O/N at 4 C; 2) 0.1 ug/well linearCGQKLVG—conjugated-BSA (SEQ ID NO:3) at 100 uL/well in carbonate coatingbuffer (pH 9.6) O/N at 4 C; or 3) 0.1 ug/well Negative-Peptide at 100uL/well in carbonate coating buffer (pH 9.6) O/N at 4 C. PrimaryAntibody: Hybridoma supernatant at 100 uL/well incubated for 1 hour at37 C with shaking. Secondary Antibody 1:10,000 Goat anti-mouseIgG/IgM(H+L)-HRP at 100 uL/well in PBS-Tween for 1 hour at 37 C withshaking. All washing steps were performed for 30 mins with PBS-Tween.The substrate TMB was added at 50 uL/well, developed in the dark andstopped with equal volume 1M HCl.

Isotyping

The hybridoma antibodies were isotyped using antibody trap experiments.Trap plates were coated with 1:10,000 Goat anti-mouse IgG/IgM(H&L)antibody at 100 uL/well carbonate coating buffer pH9.6 overnight at 4 C.No blocking step was used. Primary antibody (hybridoma supernatants) wasadded (100 ug/mL). Secondary Antibody 1:5,000 Goat anti-mouse IgGγ-HRPor 1:10,000 Goat anti-mouse IgMμ-HRP at 100 uL/well in PBS-Tween for 1hour at 37 C with shaking. All washing steps were performed for 30 minswith PBS-Tween. The substrate TMB was added at 50 uL/well, developed inthe dark and stopped with equal volume 1M HCl.

SPR Binding Assays—Primary and Secondary Screens

SPR Analysis of Antibody Binding to A-Beta Monomers and Oligomers

A-Beta Monomer and Oligomer Preparation

Recombinant A-beta40 and 42 peptides (California Peptide, Salt Lake CityUtah, USA) were dissolved in ice-cold hexafluoroisopropanol (HFIP). TheHFIP was removed by evaporation overnight and dried in a SpeedVaccentrifuge. To prepare monomers, the peptide film was reconstituted inDMSO to 5 mM, diluted further to 100 μM in dH2O and used immediately.Oligomers were prepared by diluting the 5 mM DMSO peptide solution inphenol red-free F12 medium (Life Technologies Inc., Burlington ON,Canada) to a final concentration of 100 μM and incubated for 24 hours to7 days at 4° C.

SPR Analysis All SPR measurements were performed using a MolecularAffinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg,Germany), an analytical biosensor that employs high intensity laserlight and high speed optical scanning to monitor binding interactions inreal time. The primary screening of tissue culture supernatants wasperformed using an SPR direct binding assay, whereby BSA-conjugatedpeptides, A-beta42 Monomer and A-beta42 Oligomer are covalentlyimmobilized on individual flow cells of a High Amine Capacity (HAC)sensorchip (Sierra Sensors GmbH, Hamburg, Germany) and antibodies flowedover the surface. Protein G purified mAbs were analyzed in a secondaryscreen using an SPR indirect (capture) binding assay, whereby theantibodies were captured on a protein A-derivatized sensorchip (XanTecBioanalytics GmbH, Duesseldorf, Germany) and A-beta40 Monomer, A-beta42Oligomer, soluble brain extracts and cerebrospinal fluid flowed over thesurface. The specificity of the antibodies was verified in an SPR directbinding assay by covalently immobilizing A-beta42 Monomer and A-beta42Oligomer on individual flow cells of a HAC sensorchip and flowingpurified mAbs.

SPR Analysis of Soluble Brain Extracts and CSF Samples

Soluble Brain Extract and CSF Preparation

Human brain tissues and CSFs were obtained from patients assessed at theUBC Alzheimer's and Related Disorders Clinic. Clinical diagnosis ofprobable AD is based on NINCDS-ADRDA criteria [5]. CSFs are collected inpolypropylene tubes, processed, aliquoted into 100 μL polypropylenevials, and stored at −80° C. within 1 hour after lumbar puncture.

Homogenization:

Human brain tissue samples were weighed and subsequently submersed in avolume of fresh, ice cold TBS (supplemented with EDTA-free proteaseinhibitor cocktail from Roche Diagnostics, Laval QC, Canada) such thatthe final concentration of brain tissue is 20% (w/v). Tissue ishomogenized in this buffer using a mechanical probe homogenizer (3×30sec pulses with 30 sec pauses in between, all performed on ice). TBShomogenized samples are then subjected to ultracentrifugation (70,000×gfor 90 min). Supernatants are collected, aliquoted and stored at −80° C.The protein concentration of TBS homogenates is determined using a BCAprotein assay (Pierce Biotechnology Inc, Rockford Ill., USA).

SPR Analysis

Brain extracts from 4 AD patients and 4 age-matched controls, and CSFsamples from 9 AD patients and 9 age-matched controls were pooled andanalyzed. Purified mAbs were captured on separate flow cells of aprotein A-derivatized sensor chip and diluted samples injected over thesurfaces for 180 seconds, followed by 120 seconds of dissociation inbuffer and surface regeneration. Binding responses weredouble-referenced by subtraction of mouse control IgG monoclonalantibody reference surface binding and assay buffer, and the differentgroups of samples compared

Assessing Binding or Lack thereof to A-Beta Monomers

In the primary screen of tissue culture supernatants, A-beta42 monomersand A-beta42 oligomers were used in a direct binding assay. In thesecondary screen, A-beta40 monomers and A-beta42 oligomers soluble brainextracts and CSF samples were used in an indirect (capture) bindingassay.

Primary Screen

Tissue culture supernatants were screened for the presence of antibodybinding against their cognate cyclic peptide. Each sample was dilutedand injected in duplicate over the immobilized peptide and BSA referencesurfaces for 120 seconds, followed by injection of running buffer onlyfor a 300-second dissociation phase. After every analytical cycle, thesensor chip surfaces were regenerated. Sensorgrams weredouble-referenced by subtracting out binding from the BSA referencesurfaces and blank running buffer injections, and binding responsereport points collected in the dissociation phase.

Oligomer Binding Assay

Next synthetic A-beta 42 oligomers were generated and immobilized asabove, antibody binding responses analyzed. Antibody binding responsesto A-beta 42 oligomers were compared to binding responses to cyclic.

Verifying Binding to A-Beta Oligomers

To further verify and validate A-beta42 Oligomer binding, antibodieswere covalently immobilized, followed by the injection over the surfaceof commercially-prepared stable A-beta42 Oligomers (SynAging SAS,Vandceuvre-lés-Nancy, France).

Results

ELISA testing found that the majority of hybridoma clones bound thecyclopeptide.

Next clones were tested by ELISA for their binding selectivity forcyclo- and linear-CGQKLVG (SEQ ID NO: 3) compounds. A number of clonespreferentially bound cyclo(CGQKLVG)—conjugated-BSA (SEQ ID NO: 3)compared to linear CGQKLVG—conjugated-BSA (SEQ ID NO: 3).

Isotyping revealed that the majority of clones were IgG including IgG1,IgG2a and IgG3 clones. Several IgM and IgA clones were also identified,but not pursued further.

A direct binding analysis using surface plasmon resonance was performedto screen for antibodies in tissue culture supernatants that bind to thecyclic peptide of SEQ ID NO: 3.

FIG. 16 plots the SPR direct binding assay data versus the ELISA bindingdata, and shows that there is a correlation between the SPR directbinding and ELISA results. Strong binding in SPR only occurs when thereis strong binding in ELISA.

Clones were tested for their ability to bind cyclic peptide, linearpeptide, A-beta 1-42 monomer and A-beta 1-42 oligomers prepared asdescribed above. Binding assays were performed using MASS-1 as describedabove (Direct binding assays). A number of clones were selected based onthe binding assays performed as shown in Table 1.

The selected clones were IgG mAb. Negative numbers in the primary screenare indicative of no binding (e.g. less than isotype control).

TABLE 1 305 Cyclic- Linear- A β 42 Monomer A β 42 Peptide (RU) Peptide(RU) (RU) Oligomer (RU) 2A8 333.1 −6.1 −13.5 76.6 3D8 639.7 5.7 −20.9 454C2 362.2 −4.3 −34 161.6 4C12 433.3 24.7 73.1 5D3 292.7 19.1 −12.8 47.45G1 295.5 120.5 −17.6 46.2 6E3 211 12.4 −36.6 77.5 7E9 495.4 7.7 −55.186 8H10 533.7 83.8 −32.8 68.5 9F3 304.6 36.4 −7.2 56.6 10B6 263.3 9.3−13.4 71.9 12D5 259.8 −3.2 −7 31.7 12F4 554.8 −0.8 38.7 89.2ELISA Prescreen

The ELISA prescreen of hybridoma supernatants identified clones thatshowed increased binding to the cyclic peptides compared to the linearpeptide. A proportion of the clones were reactive to KLH-epitope linkerpeptide. These were excluded from further investigation. The majority ofthe clones were determined to be of the IgG isotype using the isotypingprocedure described herein.

Direct Binding Measured by Surface Plasmon Resonance—Primary Screen

Using surface plasmon resonance the antibody clone containing tissueculture supernatants were tested for direct binding to cyclic peptide,linear peptide, A-beta oligomer and A-beta monomer.

The results for are shown in FIG. 15. Panel A shows antibody binding tocyclic peptide and to linear peptide (unstructured), for IgG clones thatare not reactive to the linker region. Panel B shows antibody binding toA-beta oligomer and A-beta monomer. A number of the clones have elevatedreactivity to the cyclic peptide and all clones have minimal or noreactivity to linear peptide, except for one. There is a generalselectivity for A-beta oligomer binding.

For select clones comparative binding profile is shown in FIG. 17. Eachclone is assessed for direct binding using surface plasmon resonanceagainst specific epitope in the context of cyclic peptide (circle),linear peptide (square), A-beta (Aβ) monomer (upright triangle), andA-beta oligomer (AβO) (upside-down triangle).

Example 9

Secondary Screen

Immunohistochemistry

Immunohistochemistry was performed on frozen human brain sections, withno fixation or antigen retrieval. In a humidified chamber, non-specificstaining was blocked by incubation with serum-free protein blockingreagent (Dako Canada Inc., Mississauga, ON, Canada) for 1 h. Thefollowing primary antibodies were used for immunostaining: mousemonoclonal isotype controls IgG1, IgG2a, and IgG2b, and anti-amyloidβ6E10, all purchased from Biolegend, and selected purified clonesreactive to the cyclopeptide. All antibodies were used at 1 μg/mL.Sections were incubated at room temperature for 1 h, and washed 3×5 minin TBS-T. Anti-Mouse IgG Horseradish Peroxidase conjugated (1:1000, ECL)was applied to sections and incubated 45 min, then washed 3×5 min inTBS-T. DAB chromogen reagent (Vector Laboratories, Burlington ON,Canada) was applied and sections rinsed with distilled water when thedesired level of target to background staining was achieved. Sectionswere counterstained with Mayer's haematoxylin, dehydrated and coverslips were applied. Slides were examined under a light microscope (ZeissAxiovert 200M, Carl Zeiss Canada, Toronto ON, Canada) and representativeimages captured at 20 and 40× magnification using a Leica DC300 digitalcamera and software (Leica Microsystems Canada Inc., Richmond Hill, ON).Images were optimized in Adobe Photoshop using Levels Auto Correction.

CSF and Brain Extracts

Human brain tissues were obtained from the University of Maryland Brainand Tissue Bank upon approval from the UBC Clinical Research EthicsBoard (C04-0595). CSFs were obtained from patients assessed at the UBCHospital Clinic for Alzheimer's and Related Disorders. The study wasapproved by the UBC Clinical Research Ethics Board, and written consentfrom the participant or legal next of kin was obtained prior tocollection of CSF samples. Clinical diagnosis of probable AD was basedon NINCDS-ADRDA criteria. CSFs were collected in polypropylene tubes,processed, aliquoted into 100 μL polypropylene vials, and stored at −80°C. within 1 hour after lumbar puncture.

Homogenization: Human brain tissue samples were weighed and subsequentlysubmersed in a volume of fresh, ice cold TBS and EDTA-free proteaseinhibitor cocktail from Roche Diagnostics (Laval QC, Canada) such thatthe final concentration of brain tissue was 20% (w/v). Tissue washomogenized in this buffer using a mechanical probe homogenizer (3×30sec pulses with 30 sec pauses in between, all performed on ice). TBShomogenized samples were then subjected to ultracentrifugation (70,000×gfor 90 min). Supernatants were collected, aliquoted and stored at −80°C. The protein concentration of TBS homogenates was determined using aBCA protein assay (Pierce Biotechnology Inc, Rockford Ill., USA).

CSF: CSF was pooled from 9 donors with AD and 9 donors without AD.Samples were analyzed by SPR using purified IgG at a concentration of 30micrograms/ml for all antibodies. Mouse IgG was used as an antibodycontrol and all experiments were repeated at least 2 times.

Positive binding in CSF and brain extracts was confirmed using antibody6E10.

SPR Analysis: 4 brain extracts from AD patients and 4 brain extractsfrom age-matched controls were pooled and analyzed. Brain samples,homogenized in TBS, included frontal cortex Brodmann area 9. Allexperiments were performed using a Molecular Affinity Screening System(MASS-1) (Sierra Sensors GmbH, Hamburg, Germany), an analyticalbiosensor that employs high intensity laser light and high speed opticalscanning to monitor binding interactions in real time as described inExample 6. Purified antibodies generated for cyclopeptides describedherein were captured on separate flow cells of a protein A-derivatizedsensor chip and diluted samples injected over the surfaces for 180seconds, followed by 120 seconds of dissociation in buffer and surfaceregeneration. Binding responses were double-referenced by subtraction ofmouse control IgG reference surface binding and assay buffer, and thedifferent groups of samples compared.

Results

CSF Brain Extracts and Immunohistochemistry

Several clones were tested for their ability to bind A-beta in CSF,soluble brain extracts and tissue samples of cavaderic AD brains areshown in Table 2. Strength of positivity in Table 2 is shown by thenumber plus signs.

Table 2 and Table 3 provide data for selected clone's bindingselectivity for oligomers over monomer measured as described herein bySPR.

IHC results are also summarized in Table 2 where “+/−” denotes stainingsimilar to or distinct from isotype control but without clear plaquemorphology.

FIG. 18 shows an example of the lack of plaque staining on fresh frozensections with clone 8H10 (62) compared to the positive plaque stainingseen with 6E10 antibody.

FIG. 19 shows, antibodies raised to the cyclopeptide comprising QKLV(SEQ ID NO: 1) bound A-beta oligomer preferentially over monomer andalso preferentially bound A-beta in brain extracts and/or CSF of ADpatients.

As shown in Tables 2, 3 and FIGS. 18 and 19, antibodies raised to thecyclopeptide comprising QKLV (SEQ ID NO: 1) included clones that boundA-beta in brain extracts and/or CSF, but did not appreciably bind tomonomers on SPR, and did not appreciably bind to plaque fibrils by IHC.

TABLE 2 Summary of binding characteristics Oligomers/ CSF Brain ExtractIHC - Plaque Clone # Monomers AD/Non-AD AD/Non-AD Staining cyclo(CGQKLVG) 305-59 + − ++ +/− (SEQ ID NO: 3) (5G1) 305-61 (7E9) − ++ − −305-62 +/− − + − (8H10) *Scoring is relative to other clones in the samesample category.

TABLE 3 A-beta Oligomer binding RU values subtracted for monomer bindingClone tested 305-62 (8H10) RU 0.4

Example 10

Synthetic Oligomer Binding

Serial 2-fold dilutions (7.8 nM to 2000 nM) of commercially-preparedsynthetic amyloid beta oligomers (SynAging SAS, Vandceuvre-lés-Nancy,were tested for binding to covalently immobilized antibodies. Resultsfor control antibody mAb6E10 is shown in FIG. 20A and mouse control IgGcontrol is shown in FIG. 20B. FIG. 20 C shows results using an antibodyraised against cyclo(CGQKLVG) (SEQ ID NO:3).

Example 11

Immunohistochemistry on Formalin Fixed Tissues

Human brain tissue was assessed using antibodies raised to cyclo CGQKLVG(SEQ ID NO: 3. The patient had been previously characterized anddiagnosed with Alzheimer's disease with a tripartite approach: (i)Bielschowsky silver method to demonstrate senile plaques andneurofibrillary tangles, (ii) Congo red to demonstrate amyloid and (iii)tau immunohistochemistry to demonstrate tangles and to confirm thesenile plaques are “neuritic”. This tissue was used to test plaquereactivity of selected monoclonal antibody clones. The brain tissueswere fixed in 10% buffered formalin for several days and paraffinprocessed in the Sakura VIP tissue processors. Tissue sections wereprobed with 1 μg/ml of antibody with and without microwave antigenretrieval (AR). The pan-amyloid beta reactive antibody 6E10 was includedalong with selected antibody clones as a positive control. Antibodieswere diluted in Antibody Diluent (Ventana), color was developed withOptiView DAB (Ventana). The staining was performed on the VentanaBenchmark XT IHC stainer. Images were obtained with an Olympus BX45microscope. Images were analyzed blind by a professional pathologistwith expertise in neuropathology.

As shown in Table 4 below, using fixed tissue, the tested antibodieswere negative for specific staining of senile plaque amyloid with orwithout antigen retrieval. 6E10 was used as the positive control.

TABLE 4 Convincing evidence of specific staining of senile plaqueamyloid Epitope Antibodies to test Without AR Plus AR 305 59 (5G1)possible weak staining Neg 61 (7E9) Neg Neg 62 (8H10) Neg Neg Positive6E10 Strongly positive Strongly positive Control

Example 12

Inhibition of Oligomer Propagation

The biological functionality of antibodies was tested in vitro byexamining their effects on Amyloid Beta (Aβ) aggregation using theThioflavin T (ThT) binding assay. Aβ aggregation is induced by andpropagated through nuclei of preformed small Aβ oligomers, and thecomplete process from monomeric Aβ to soluble oligomers to insolublefibrils is accompanied by concomitantly increasing beta sheet formation.This can be monitored by ThT, a benzothiazole salt, whose excitation andemission maxima shifts from 385 to 450 nm and from 445 to 482 nmrespectively when bound to beta sheet-rich structures and resulting inincreased fluorescence Briefly, Aβ 1-42 (Bachem Americas Inc., Torrance,Calif.) was solubilized, sonicated, diluted in Tris-EDTA buffer (pH 7.4)and added to wells of a black 96-well microtitre plate (Greiner Bio-One,Monroe, N.C.) to which equal volumes of cyclopeptide raised antibody orirrelevant mouse IgG antibody isotype controls were added, resulting ina 1:5 molar ratio of Aβ1-42 peptide to antibody. ThT was added andplates incubated at room temperature for 24 hours, with ThT fluorescencemeasurements (excitation at 440 nm, emission at 486 nm) recorded everyhour using a Wallac Victor3v 1420 Multilabel Counter (PerkinElmer,Waltham, Mass.). Fluorescent readings from background buffer weresubtracted from all wells, and readings from antibody only wells werefurther subtracted from the corresponding wells. As shown in FIG. 21,Aβ42 aggregation, as monitored by ThT fluorescence, demonstrated asigmoidal shape characterized by an initial lag phase with minimalfluorescence, an exponential phase with a rapid increase in fluorescenceand finally a plateau phase during which the Aβ molecular species are atequilibrium and during which there is no increase in fluorescence.Co-incubation of Aβ42 with an irrelevant mouse antibody did not have anysignificant effect on the aggregation process. In contrast,co-incubation of Aβ42 with the test antibodies completely inhibited allphases of the aggregation process. Results obtained with antibody clones61, 62 and 64 are shown in FIG. 21. As the ThT aggregation assay mimicsthe in vivo biophysical/biochemical stages of Aβ propagation andaggregation from monomers, oligomers, protofibrils and fibrils that ispivotal in AD pathogenesis, the antibodies raised to cyclo CGQKLVG (SEQID NO: 3) demonstrate the potential to completely abrogate this process.Isotype control performed using mouse IgG control showed no inhibition

Example 13

Achieving the optimal profile for Alzheimer's immunotherapy: Rationalgeneration of antibodies specific for toxic A-beta oligomers

Objective: Generate antibodies specific for toxic amyloid-β oligomers(AβO)

Background: Current evidence suggests that propagating prion-likestrains of AβO, as opposed to monomers and fibrils, are preferentiallytoxic to neurons and trigger tau pathology in Alzheimer's disease (AD).In addition, dose-limiting adverse effects have been associated with Aβfibril recognition in clinical trials. These observations suggest thatspecific neutralization of toxic AβOs may be desirable for safety andefficacy.

Design/Methods: Computational simulations were employed as describedherein, using molecular dynamics with standardized force-fields toperturb atomic-level structures of Aβ fibrils deposited in the ProteinData Base. It was hypothesized that weakly-stable regions are likely tobe exposed in nascent protofibrils or oligomers. Clustering analysis,curvature, exposure to solvent, solubility, dihedral angle distribution,and Ramachandran angle distributions were all used to characterize theconformational properties of predicted epitopes, which quantifydifferences in the antigenic profile when presented in the context ofthe oligomer vs the monomer or fibril. The candidate peptide epitopeswere synthesized in a cyclic format that may mimic regional AβOconformation, conjugated to a carrier protein, and used to generatemonoclonal antibodies in mice. Purified antibodies were screened by SPRand immunohistochemistry.

Results

Sixty-six IgG clones against 5 predicted epitopes were selected forpurification based on their ability to recognize the cognate structuredpeptide and synthetic AβO, with little or no binding to unstructuredpeptide, linker peptide, or Aβ monomers. Additional screening identifiedantibodies that preferentially bound to native soluble AβO in CSF andbrain extracts of AD patients compared to controls. Immunohistochemicalanalysis of AD brain allowed for selection of antibody clones that donot react with plaque.

Conclusion: Computationally identified AβO epitopes allowed for thegeneration of antibodies with the desired target profile of selectivebinding to native AD AβOs with no significant cross-reactivity tomonomers or fibrils.

Example 14

Toxicity Inhibition Assay

The inhibition of toxicity of A-beta42 oligomers by antibodies raised tothe cyclopeptide can be tested in a rat primary cortical neuron assay.

Antibody and control IgG are each adjusted to a concentration such as 2mg/mL. Various molar ratios of A-beta oligomer and antibody are testedalong with a vehicle control, A-beta oligomer alone and a positivecontrol such as the neuroprotective peptide humanin HNG.

An exemplary set up is shown in Table 5.

Following preincubation for 10 minutes at room temperature, the volumeis adjusted to 840 microlitres with culture medium. The solution isincubated for 5 min at 37 C. The solution is then added directly to theprimary cortical neurons and cells are incubated for 24 h. Cellviability can be determined using the MTT assay.

TABLE 5 AβO/AB AβO AβO AB AB Medium Final volume molar ratio (μL) (μM)(μM) (μL) (μL) (μL) 5/1 1.68 4.2 0.84 12.73 185.6 200 1/1 1.68 4.2 4.2063.64 134.7 200 1/2 1.68 4.2 8.4 127.27 71.1 200 AβO working solution:2.2 mg/mL-500 μM CTRL vehicle: 1.68 μL of oligomer buffer + 127.3 μLPBS + 711 μL culture medium CTRL AβO: 1.68 μL of AβO + 127.3 μL PBS +711 μL culture medium CTRL HNG: 1.68 μL of AβO + 8.4 μL HNG (100 nMfinal) + 127.3 μL PBS + 702.6 μL culture medium

This test was conducted using 305 antibody clone 62. The antibody aloneshowed no toxicity (FIG. 22A). Dosage-independent inhibition of A-betaoligomer toxicity was observed for all concentrations of antibody tooligomer: 1:5, 1:1 and 2:1 (FIG. 22B).

Example 15

In Vivo Toxicity Inhibition Assay

The inhibition of toxicity of A-beta42 oligomers by antibodies raised tothe cyclopeptide can be tested in vivo in mouse behavioral assays.

The antibody and an isotype control are each pre-mixed with A-beta42oligomers at 2 or more different molar ratios prior tointracerebroventricular (ICV) injection into mice. Control groupsinclude mice injected with vehicle alone, oligomers alone, antibodyalone, and a positive control such as the neuroprotective peptidehumanin. Alternatively, the antibodies can be administered systemicallyprior to, during, and/or after ICV injection of the oligomers. Startingapproximately 4-7 days post ICV injection of oligomers, cognition isassessed in behavioral assays of learning and memory such as the mousespatial recognition test (SRT), Y-Maze assay, Morris water maze modeland novel object recognition model (NOR).

The mouse spatial recognition test (SRT) assesses topographical memory,a measure of hippocampal function (SynAging). The model uses atwo-chamber apparatus, in which the chambers differ in shape, patternand color (i.e. topographical difference). The chambers are connected bya clear Plexiglass corridor. Individual mice are first placed in theapparatus for a 5 min exploration phase where access to only one of thechambers is allowed. Mice are then returned to their home cage for 30min and are placed back in the apparatus for a 5 min “choice” phaseduring which they have access to both chambers. Mice with normalcognitive function remember the previously explored chamber and spendmore time in the novel chamber. A discrimination index (DI) iscalculated as follows: DI=(TN−TF)/(TN+TF), in which TN is the amount oftime spent in the novel chamber and TF is the amount of time spent inthe familiar chamber. Toxic A-beta oligomers cause a decrease in DIwhich can be partially rescued by the humanin positive control.Performance of this assay at different time points post ICV injectioncan be used to evaluate the potential of antibodies raised to thecyclopeptide to inhibit A-beta oligomer toxicity in vivo.

The Y-maze assay (SynAging) is a test of spatial working memory which ismainly mediated by the prefrontal cortex (working memory) and thehippocampus (spatial component). Mice are placed in a Y-shaped mazewhere they can explore 2 arms. Mice with intact short-term memory willalternate between the 2 arms in successive trials. Mice injected ICVwith toxic A-beta oligomers are cognitively impaired and show randombehavior with alternation close to a random value of 50% (versus ˜70% innormal animals). This impairment is partially or completely reversed bythe cholinesterase inhibitor donepezil (Aricept) or humanin,respectively. This assay provides another in vivo assessment of theprotective activity of test antibodies against A-beta oligomer toxicity.

The Morris water maze is another widely accepted cognition model,investigating spatial learning and long-term topographical memory,largely dependent on hippocampal function (SynAging). Mice are trainedto find a platform hidden under an opaque water surface in multipletrials. Their learning performance in recalling the platform location isbased on visual clues and video recorded. Their learning speed, which isthe steadily reduced time from their release into the water untilfinding the platform, is measured over multiple days. Cognitively normalmice require less and less time to find the platform on successive days(learning). For analyzing long-term memory, the test is repeatedmultiple days after training: the platform is taken away and the numberof crossings over the former platform location, or the time of the firstcrossing, are used as measures to evaluate long-term memory. Miceinjected ICV with toxic A-beta oligomers show deficits in both learningand long-term memory and provide a model for evaluating the protectiveactivity of test antibodies.

The Novel Object Recognition (NOR) model utilizes the normal behavior ofrodents to investigate novel objects for a significantly longer timethan known objects, largely dependent on perirhinal cortex function(SynAging). Mice or rats are allowed to explore two identical objects inthe acquisition trial. Following a short inter-trial interval, one ofthe objects is replaced by a novel object. The animals are returned tothe arena and the time spent actively exploring each object is recorded.Normal rodents recall the familiar object and will spend significantlymore time exploring the novel object. In contrast, A-betaoligomer-treated rodents exhibit clear cognitive impairment and willspend a similar amount of time investigating both the ‘familiar’ and‘novel’ object. This can be transiently reversed with known clinicalcognitive enhancers (e.g. donepezil). The NOR assay can be performedmultiple times in longitudinal studies to assess the potential cognitivebenefit of test antibodies.

In addition to behavioral assays, brain tissue can be collected andanalyzed for levels of synaptic markers (PSD95, SNAP25, synaptophysin)and inflammation markers (IL-1-beta). Mice are sacrificed at ˜14 dayspost-ICV injection of oligomers and perfused with saline. Hippocampi arecollected, snap frozen and stored at −80° C. until analyzed. Proteinconcentrations of homogenized samples are determined by BCA.Concentration of synaptic markers are determined using ELISA kits(Cloud-Clone Corp, USA). Typically, synaptic markers are reduced by25-30% in mice injected with A-beta oligomers and restored to 90-100% bythe humanin positive control. Concentrations of the IL-1-betainflammatory markers are increased approximately 3-fold in mice injectedwith A-beta oligomers and this increase is largely prevented by humanin.These assays provide another measure of the protective activity of testantibodies at the molecular level.

Example 16

In vivo Propagation Inhibition Assay

In vivo propagation of A-beta toxic oligomers and associated pathologycan be studied in various rodent models of Alzheimer's disease (AD). Forexample, mice transgenic for human APP (e.g. APP23 mice) or human APPand PSEN1 (APPPS1 mice) express elevated levels of A-beta and exhibitgradual amyloid deposition with age accompanied by inflammation andneuronal damage. Intracerebral inoculation of oligomer-containing brainextracts can significantly accelerate this process13, 14). These modelsprovide a system to study inhibition of A-beta oligomer propagation bytest antibodies administered intracerebrally or systemically.

Example 17

CDR Sequencing-305-7E9.1

305-7E9.1 which was determined to have an IgG3 heavy chain and a kappalight chain was selected for CDR and variable regions of the heavy andlight chains.

RT-PCR was carried out using 5′ RACE and gene specific reverse primerswhich amplify the appropriate mouse immunoglobulin heavy chain(IgG1/IgG3/IgG2A) and light chain (kappa) variable region sequences.

The specific bands were excised and cloned into pCR-Blunt II-TOPO vectorfor sequencing, and the constructs were transformed into E. coli

At least 8 colonies of each chain were picked & PCR screened for thepresence of amplified regions prior to sequencing. Selected PCR positiveclones were sequenced.

The CDR sequences are in Table 6. The consensus DNA sequence and proteinsequences of the variable portion of the heavy and light chain areprovided in Table 7.

TABLE 6 Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GYTFTDYE 11 CDR-H2IDPETGDT 12 CDR-H3 TSPIYYDYDWFAY 13 Light CDR-L1 QSLLNNRTRKNY 14 CDR-L2WAS 15 CDR-L3 KQSYNLRT 16

TABLE 7Consensus DNA sequence and translated protein sequences of the variable region.The complementarity determining regions (CDRs) are underlined according to IMTG/LIGM-DB. Isotype Consensus DNA Sequence Protein sequence IgG3ATGGAATGGAGCTGGGTCTTTCTCTTCCTCCTGTCAGTAATTGCAGGTG MEWSWVFLFLLSVIAGVSEQ ID NO: TCCAATCCCAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCQSQVQLQQSGAELVRPG 17, 18 CTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTA ASVTLSCKAS GYTFTDYE CTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGG MHWVKQTPVHGLEWIG AATGGATTGGAGCTATTGATCCTGAAACTGGTGATACT GCCTACAATC A IDPETGDT AYNQEFKGKAGGAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCA ATLTADKSSSTAYMELRSLCAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCT TSEDSAVYYC TSPIYYDYDATTACTGT ACAAGCCCCATCTACTATGATTACGACTGGTTTGCTTAC TG WFAY WGHGTLVTVSAATGGGCCACGGGACTCTGGTCACTGTCTCTGCAGCTACAACAACAGCCCC TTAPS ATCT KappaATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTATCTG MDSQAQVLILLLLWVSGTSEQ ID NO: GTACCTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTCGDIVMSQSPSSLAVSAG 19, 20 GTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTC EKVTMSCKSS QSLLNNRT TGCTCAACAATAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAG RKNY LAWYQQKPGQSPKAAACCAGGGCAGTCTCCTAAACTGCTGATCTAC TGGGCATCC ACTAGG LLIY WAS TRESGVPDRFTGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGAT GSGSGTDFTLTISSVQAEDTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATT LAVYYC KQSYNLRT FGGGACTGC AAGCAATCTTATAATCTTCGGACG TTCGGTGGAGGCACCAAGC TKLEIKRADATGGAAATCAAACGGGCTGATGCT

Example 9

CDR sequencing—305-8H10

305-8H10 which was determined to have an IgG1 heavy chain and a kappalight chain was selected for sequencing CDR and variable regions of theheavy and light chains.

RT-PCR was carried out using 5′ RACE and gene specific reverse primerswhich amplify the appropriate mouse immunoglobulin heavy chain(IgG1/IgG3/IgG2A) and light chain (kappa) variable region sequences.

The specific bands were excised and cloned into pCR-Blunt II-TOPO vectorfor sequencing, and the constructs were transformed into E. coli

At least 8 colonies of each chain were picked & PCR screened for thepresence of amplified regions prior to sequencing. Selected PCR positiveclones were sequenced.

The CDR sequences are in Table 8. The consensus DNA sequence and proteinsequences of the variable portion of the heavy and light chain areprovided in Table 9.

TABLE 8 Chain CDR Sequence SEQ ID NO Heavy CDR-H1 GFSLSTSGMG 21 CDR-H2IWWDDDK 22 CDR-H3 ARSITTVVATPFDY 23 Light CDR-L1 QNVRSA 24 CDR-L2 LAS 25CDR-L3 LQHWNSPFT 26

TABLE 9Consensus DNA sequence and translated protein sequences of the variable region.The complementarity determining regions (CDRs) are underlined according toIMTG/LIGM-DB. Isotype Consensus DNA Sequence Protein sequence IgG1ATGGACAGGCTTACTTCTTCATTCCTGCTGCTGATTGTCCCTGCA MDRLTSSFLLLIVPAYSEQ ID NO: TATGTCTTGTCCCAAGTTACTCTAAAAGAGTCTGGCCCTGGGATAVLSQVTLKESGPGILK 27, 28 TTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCT GGGPSQTLSLTCSFSG FSL TTTTCACTGAGCACTTCTGGTATGGGT GTAGGCTGGATTCGTCAG STSGMGVGWIRQPSGK CCTTCAGGGAAGGGTCTGGAGTGGCTGGCACAC ATTTGGTGGGAT GLEWLAHIWWDDDK YY GATGATAAG TACTATAACCCATCCCTGAAGAGCCAGCTCACAATCNPSLKSQLTISKDTSR TCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGATCACCAGTNQVFLKITSVDTADTA GTGGACACTGCAGATACTGCCACTTACTACTGT GCTCGAAGTATT TYYCARSITTVVATPF ACTACGGTAGTAGCTACGCCCTTTGACTAC TGGGGCCAAGGCACC DYWGQGTTLTVSSAKT ACTCTCACAGTCTCCTCAGCCAAAACGACAC T KappaATGGGCATCAAGATGGAGTTTCAGACCCAGGTCTTTGTATTCGTG MGIKMEFQTQVFVFVLSEQ ID NO: TTGCTCTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGLWLSGVDGDIVMTQSQ 29, 30 TCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCKFMSTSVGDRVSITCK ACCTGCAAGGCCAGT CAGAATGTTCGTTCTGCT GTAGCCTGGTAT ASQNVRSA VAWYQQKP CAACAGAAACCAGGGCAGTCTCCTAAAGCACTGATTTAC CTGGCA GQSPKALIYLAS NRHT TCC AACCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGA GVPDRFTGSGSGTDFTTCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCATTCTGAA LTISNVHSEDLTDYFCGACCTGACAGATTATTTCTGT CTGCAACATTGGAATTCTCCGTTC LQHWNSPFT FGGGTKL ACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCT EIKRADA

TABLE 10 A-beta Sequences and A beta sequences with linkerQKLV (SEQ ID NO: 1) HQKLV (SEQ ID NO: 2)CGQKLVG, cycloCGQKLVG (SEQ ID NO: 3) GQKLV (SEQ ID NO: 4)GQKLVG (SEQ ID NO: 5) GGQKLVG (SEQ ID NO: 6) GQKLVGG (SEQ ID NO: 7)CGQKLVGC (SEQ ID NO: 8) HQKLVF (SEQ ID NO: 9) QKLVF (SEQ ID NO: 10)

TABLE 11 Full A-beta 1-42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(SEQ ID NO: 31)

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

The scope of the claims should not be limited by the preferredembodiments and examples, but should be given the broadestinterpretation consistent with the description as a whole.

CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

-   [1] SCIENTIFIC REPORTS|5: 9649|DOI: 10.1038/srep09649-   [2] Vincent J. Hilser and Ernesto Freire. Structure-based    calculation of the equilibrium folding pathway of proteins.    correlation with hydrogen exchange protection factors. J. Mol.    Biol., 262:756-772, 1996. The COREX approach.-   [3] Samuel I. A. Cohen, Sara Linse, Leila M. Luheshi, Erik    Hellstrand, Duncan A. White, Luke Rajah, Daniel E. Otzen, Michele    Vendruscolo, Christopher M. Dobson, and Tuomas P. J. Knowles.    Proliferation of amyloid-β42 aggregates occurs through a secondary    nucleation mechanism. Proc. Natl. I Acad. Sci. USA,    110(24):9758-9763, 2013.-   [4] Pietro Sormanni, Francesco A. Aprile, and Michele Vendruscolo.    The camsol method of rational design of protein mutants with    enhanced solubility. Journal of Molecular Biology, 427(2):478-490,    2015.-   [5] Deborah Blacker, MD, ScD; Marilyn S. Albert, PhD; Susan S.    Bassett, PhD; Rodney C. P. Go, PhD; Lindy E. Harrell, MD, PhD;    Marshai F. Folstein, MD Reliability and Validity of NINCDS-ADRDA    Criteria for Alzheimer's Disease The National Institute of Mental    Health Genetics Initiative. Arch Neurol. 1994; 51(12):1198-1204.    doi:10.1001/archneur.1994.00540240042014.-   [6] Hamley, I. W. PEG-Peptide Conjugates 2014; 15, 1543-1559;    dx.doi.org/10.1021/bm500246w-   [7] Roberts, M J et al Chemistry for peptide and protein PEGylation    64: 116-127.

The invention claimed is:
 1. An isolated antibody or antigen-bindingfragment thereof that specifically binds to a cyclic A-beta peptidecomprising the amino acid sequence of SEQ ID NO: 3, the antibody orantigen-binding fragment thereof comprising a light chain variableregion and a heavy chain variable region, the heavy chain variableregion comprising complementarity determining regions CDR-H1, CDR-H2 andCDR-H3, the light chain variable region comprising complementaritydetermining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acidsequences of said CDRs comprising the sequences of either: A. CDR-H1GYTFTDYE (SEQ ID NO: 11) CDR-H2 IDPETGDT (SEQ ID NO: 12) CDR-H3TSPIYYDYDWFAY (SEQ ID NO: 13) CDR-L1 QSLLNNRTRKNY (SEQ ID NO: 14) CDR-L2WAS (SEQ ID NO: 15) and DR-L3 KQSYNLRT (SEQ ID NO: 16); or B. CDR-H1GFSLSTSGMG (SEQ ID NO: 21) CDR-H2 IWWDDDK (SEQ ID NO: 22) CDR-H3ARSITTVVATPFDY (SEQ ID NO: 23) CDR-L1 QNVRSA (SEQ ID NO: 24) CDR-L2 LAS(SEQ ID NO: 25) and CDR-L3 LQHWNSPFT (SEQ ID NO: 26).
 2. The isolatedantibody or antigen-binding fragment thereof of claim 1, wherein theheavy chain variable region of the antibody A according to claim 1comprises the amino acid sequence of SEQ ID NO: 18 and the light chainvariable region of the antibody A according to claim 1 comprises theamino acid sequence of SEQ ID NO:
 20. 3. The isolated antibody orantigen-binding fragment thereof of claim 1, wherein the heavy chainvariable region of the antibody B according to claim 1 comprises theamino acid sequence of SEQ ID NO: 28 and the light chain variable regionof the antibody B according to claim 1 comprises the amino acid sequenceof SEQ ID NO:30.
 4. The antibody or antigen-binding fragment thereof ofclaim 1, wherein the light chain variable region and the heavy chainvariable region of the antibody A or B according to claim 1 are fused.5. The antibody or antigen-binding fragment thereof of claim 1, whereinthe antibody is monoclonal or humanized.
 6. The antibody orantigen-binding fragment thereof of claim 1, wherein the antigen-bindingfragment is a Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, nanobody,minibody, diabody of the antibody according to claim 1 or a dimer ormultimer thereof.
 7. An immunoconjugate comprising the antibody orantigen-binding fragment thereof of claim 1 and a detectable label orcytotoxic agent.
 8. The immunoconjugate of claim 7, wherein thedetectable label comprises a positron emitting radionuclide.
 9. Acomposition comprising the antibody or antigen-binding fragment thereofof claim 1, or the immunoconjugate of claim 7, optionally with adiluent.